Engineered retrons and methods of use

ABSTRACT

Disclosed are engineered retrons and methods of use such as to modify the genome of a host (e.g., mammalian) cell by delivering the engineered retron or the encoded ncRNA in vitro or in vivo to the host (e.g., mammalian) cell.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 63/301,936, filed Jan. 21, 2022, U.S. Provisional Application Ser. No. 63/370,880, filed Aug. 9, 2022, and U.S. Provisional Application Ser. No. 63/373,545, filed Aug. 25, 2022, each of which are incorporated herein by reference in their entireties.

SEQUENCE LISTING

This application contains a sequence listing filed in electronic form in eXtensible Markup Language (XML) format entitled Replacement RTX004_SL.xml, created on Mar. 31, 2023 and having a size of 32,261,692 bytes. The content of the sequence listing is incorporated herein in its entirety.

TECHNICAL FIELD

The present disclosure generally relates to systems, methods and compositions used for precise genome editing, including nucleic acid insertions, replacements, and deletions at targeted and precise genome sites, wherein said systems, methods, and compositions are based on novel and/or engineered retrons.

BACKGROUND OF THE INVENTION

Precise genome editing by programmable nucleases (e.g., RNA-guided nucleases (e.g., CRISPR nucleases), zinc-finger nucleases (ZFN), and transcription activator-like effector nucleases (TALENS)) typically relies on homology-directed repair (HDR) and the presence of a donor DNA template at the site of a double-strand break (DSB) induced by the programmable nuclease. It is generally accepted that a limiting step for HDR-dependent precise genome editing is the delivery of donor DNA template to the nuclease-induced DSB (e.g., see Ling et al., “Improving the efficiency of precise genome editing with site-specific Cas9-oligonucleotide conjugates,” Science Advances, 2020, Vol. 6, No. 15, pp. 1-8). Various methods aimed at boosting the efficiency of HDR-dependent editing have been reported, many of which involve the physical tethering of the DNA donor to a component of the precise editing system. Exemplary methods have been discussed in: K. Lee et al., “Synthetically modified guide RNA and donor DNA are a versatile platform for CRISPR-Cas9 engineering,” eLife 6, e25312 (2017); J. Carlson-Stevermer et al., “Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing,” Nat. Commun. 8, 1711 (2017); N. Savic, et al., “Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair,” eLife 7, e33761 (2018); and E. J. Aird et al., “Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template.,” Commun. Biol. 1, 54 (2018), each of which are incorporated herein by reference. Despite these efforts, efficiency of HDR-dependent precise editing remains unsatisfactory.

Retrons are defined by their unique ability to produce an unusual satellite DNA known as msDNA (multicopy single-stranded DNA). DNA encoding retrons includes a reverse trancriptase (RT)-coding gene (ret) and a nucleic acid sequence encoding the non-coding RNA (ncRNA), which contains two contiguous and inverted non-coding sequences referred to as the msr and msd. The ret gene and the non-coding RNA (including the msr and msd) are transcribed as a single RNA transcript, which becomes folded into a specific secondary structure following post-transcriptional processing. Once translated, the RT binds the RNA template downstream from the msd locus, initiating reverse transcription of the RNA towards its 5′ end, assisted by the 2′OH group present in a conserved branching guanosine residue that acts as a primer. Reverse transcription halts before reaching the msr locus, and the resulting DNA, the msDNA, remains covalently attached to the RNA template via a 2′-5′ phosphodiester bond and base-pairing between the 3′ ends of the msDNA and the RNA template. The external regions, at the 5′ and 3′ ends of the msd/msr transcript (a1 and a2, respectively) are complementary and can hybridize, leaving the structures located in the msr and msd regions in internal positions (see FIG. 1A). The msr locus, which is not reverse transcribed, forms one to three short stem-loops of variable size, ranging from 3 to 10 base pairs, whereas the msd locus folds into a single/double long hairpin with a highly variable long stem of 10-50 bp in length that is also present in the final msDNA form.

It has recently been reported that retrons may be utilized as a means to provide donor DNA template for HDR-dependent genome editing (e.g., see Lopez et al., “Precise genome editing across kingdoms of life using retron-derived DNA,” Nature Chemical Biology, Dec. 12, 2021, 18, pages 199-206 (2022)), however, producing sufficient levels of donor DNA template intracellularly to sufficiently support efficient HDR-dependent editing remains a significant challenge. Improved retron-based genome modification systems are highly desirous in the art.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides recombinant retrons comprising one or more genetic modifications which improves the functionality and/or properties of a retron. Such genetic modifications can include a mutation, insertion, deletion, inversion, replacement, substitution, or translocation of one or more contiguous or non-contiguous nucleobases in a nucleic acid molecule encoding a retron or a component of a retron, such as an ncRNA or a reverse transcriptase. In various aspects, the retron that becomes modified with the one or more genetic modifications (i.e., the “pre-modified” or “unmodified” retron or retron component) is a naturally occurring retron or retron component (e.g., naturally occurring ncRNA of Table A or RT) ability to facilitate homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentrations or amounts of msDNA comprising a DNA donor template. In particular embodiments, the recombinant retrons are based on and/or derived from a naturally-occurring retron, such as any retron-related sequence provided by Table X (the introduction of the one or more genetic modifications into a set of 7257 previously unknown retrons discovered through computational methods described herein (e.g., see Examples). In other embodiments, the recombinant retrons are based on introducing the one or more genetic modifications into previously available retron sequences (e.g., the “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classifcation of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 Dec. 2020, Pages 12632-12647” (incorporated herein by reference) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template.

In another aspect, the present disclosure further provides nucleic acid molecules encoding the recombinant retrons and/or recombinant retron components (e.g., a recombinant ncRNA and/or a recombinant retron RT). In still another aspect, the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nucleases (e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs, and TALENS), and guide RNAs (in the case where RNA-guide nucleases are used in said genome editing systems). In a further aspect, the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems. In yet another aspect, the disclosure provides vectors for transferring and/or expressing said genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions. In still another aspect, the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles. Depending on the delivery system employed, the retron-based genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combinations of approaches for delivering the components of the herein disclosed retron-based genome editing systems may be employed. In one embodiment, each of the components of the retron-based genome editing system is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease). In yet another aspect, the disclosure provides methods for genome editing by introducing a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site, thereby resulting in an edit at the target edit. In other aspects, the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems. The disclosure also provides methods of making the recombinant retrons, retron-based genome modification systems, vectors, compositions and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and/or modification systems.

In an embodiment, this disclosure or the inventions herein provide a gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo; the RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retron reverse transcriptase, (b) an engineered retron ncRNA, and (c) guide RNA for the programmable nuclease; and each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c); whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c).

In an embodiment, in the gene editing system, (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the retron reverse transcriptase.

In an embodiment, in the gene editing system, (a)(i) and (a)(ii) are encoded and expressed as a fusion protein.

In an embodiment, in the gene editing system (a)(i) and (a)(ii) are encoded and expressed as a fusion protein and the fusion protein comprises the C-terminal end of the nucleic acid programmable nuclease fused to the N-terminal end of the retron reverse transcriptase (nuclease:RT fusion); or the fusion protein comprises the N-terminal end of the nucleic acid programmable nuclease fused to the C-terminal end of the retron reverse transcriptase (RT:nuclease fusion).

In an embodiment, in the gene editing system, (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the retron reverse transcriptase.

In an embodiment, in the gene editing system, (c) is separate from (a)(i), (a)(ii) and (b) or is provided in trans.

In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis.

In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 5′ end of the retron ncRNA.

In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the guide RNA is fused to the 3′ end of the retron ncRNA.

In an embodiment, in the gene editing system, (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis and the engineered ncRNA comprises a first guide RNA fused to the 5′ end of the retron ncRNA, and a second guide RNA fused to the 3′ end of the retron ncRNA, and the first and second guide RNAs target different sequences. Thus, on a broader scale, in an embodiment, in the gene editing system, (c) guide RNA for the programmable nuclease, can comprise one or more guides that target the same or different target sequences. Such guide RNA(s) in an embodiment, can be single guide RNA(s) or sgRNA(s); for instance, when the nucleic acid programmable nuclease comprises a Cas9.

In an embodiment, in the gene editing system, the one or more delivery vehicles comprise a liposome or a lipid nanoparticle (LNP).

In an embodiment, in the gene editing system, (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retron reverse transcriptase, and (b) the engineered retron ncRNA, are in the same delivery vehicle.

In an embodiment, in the gene editing system, (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retron reverse transcriptase, and (b) the engineered retron ncRNA, are in separate delivery vehicles.

In an embodiment, in the gene editing system, the nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery vehicle.

In an embodiment, in the gene editing system, the nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles.

In an embodiment, in the gene editing system, the engineered retron ncRNA includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, and wherein the donor polynucleotide is flanked by a 5′ homology arm that hybridizes to a sequence 5′ to the target sequence and a 3′ homology arm that hybridizes to a sequence 3′ to the target sequence. In an embodiment, the donor polynucleotide can be heterologous to the cell. In an embodiment, the donor polynucleotide can be endogenous to the cell; for instance, the cell can contain a sequence that is typical for those in a population having a disease state and the donor polynucleotide can be a sequence that is typical for those in the population not having a non-disease state (e.g., the donor can be for a genetic correction or repair of a cell to modify the cell from having a mutation or modification that gives rise to a disease state to having a sequence typical of not having the disease state). Such can be done in an animal cell, or a mammalian cell (e.g., a primate, a non-human primate, or a domesticated mammal such as a cat or dog or horse) or a human cell; for instance to correct, address, treat, mitigate a genetic condition in the animal, mammal, domesticated mammal, cat, dog, horse or human. Such can be done in plant cells to introduce mutations that give rise to favorable phenotypic characteristics such as disease resistance or other favorable plant trait(s).

In an embodiment, in the gene editing system, the nucleic acid programmable nuclease comprises a Cas9 nuclease, a TnpB nuclease, or a Cas12a nuclease.

In an embodiment, in the gene editing system, the engineered retron ncRNA comprises: A) a pre-msr sequence having a first complementary region of the retron ncRNA; B) an msr sequence including an msr stem-loop structure; C) an msd sequence including an msd stem-loop structure and a sequence of interest, wherein said msd sequence templates a single strand DNA product (RT-DNA) in the presence of the retron reverse transcriptase; and D) a post-msd sequence having a second complementary region, wherein the first and second complementary regions form an a1/a2 duplex region of the retron ncRNA, wherein the msr stem-loop structure, the msd stem-loop structure, or the a1/a2 duplex comprise a modification which result in increased editing efficiency in the presence of a nucleic acid programmable nuclease that associates with the one or more guide RNAs, and wherein optionally one or more of the guide RNAs of (c) are coupled to the pre-msr sequence, the post-msd sequence, or both the pre-msr sequence and the post-msd sequence. In such an embodiment where the engineered retron ncRNA comprises A), B), C) and D), wherein the sequence of interest can encode a donor polynucleotide comprising an intended edit to be integrated at a target sequence of a cell, wherein the donor polynucleotide is flanked by a 5′ homology arm that hybridizes to a sequence 5′ to the target sequence and a 3′ homology arm that hybridizes to a sequence 3′ to the target sequence. In such an embodiment where the engineered retron ncRNA comprises A), B), C) and D) (either with the sequence of interest encoding a donor polynucleotide or simply being a sequence of interest), the ncRNA has a nucleotide sequence of Table B, or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% sequence identity with a sequence from Table B. The donor polynucleotide can be heterologous to a cell. Alternatively, the donor polynucleotide can be endogenous to the cell. For instance, the cell can contain a sequence that is typical for those in a population having a disease state and the donor polynucleotide can be a sequence that is typical for those in the population not having a non-disease state (e.g., the donor can be for a genetic correction or repair of a cell to modify the cell from having a mutation or modification that gives rise to a disease state to having a sequence typical of not having the disease state).

In an embodiment of the gene editing system, the gene editing system can comprise any combination(s) of the foregoing embodiments of the gene editing system.

In an embodiment, this disclosure or the inventions herein provide a cell, such as an isolated cell comprising the gene editing system disclosed herein, such as in any of the foregoing paragraphs. In an embodiment the cell, e.g., isolated cell, can be a eukaryotic cell. In an embodiment the eukaryotic cell can be a plant cell or an animal cell or a mammalian cell, e.g., an isolated plant cell or an isolated animal cell or an isolated mammalian cell. In an embodiment, the mammalian cell, e.g., an isolated mammalian cell, can be a human cell. In an embodiment, the cell can be a prokaryotic cell, e.g., a bacterial cell. In such an embodiment where the cell is a bacterial cell, the donor polynucleotide can code for antibiotic susceptibility; and thus, the invention can involve a means for addressing antibiotic resistant bacteria by rendering such bacteria susceptible to antibiotics (and a subject to whom the gene editing system is administered can also then receive antibiotics to which the bacteria are rendered susceptible by the gene editing system).

In an embodiment, this disclosure or the inventions herein provide a composition comprising: a) the gene editing system disclosed herein, such as in any of the foregoing paragraphs; and b) a pharmaceutically or veterinarily acceptable carrier. In an embodiment, in the composition the delivery vehicle can comprise a lipid nanoparticle comprising: a) one or more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids. In an embodiment, in the composition the one or more ionizable lipids comprises an ionizable lipid set forth in Table 2.

In an embodiment, this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in modifying a cell or genetically modifying a cell, e.g., a eukaryotic or a prokaryotic cell and/or an animal cell and/or a mammalian and/or a human cell and/or a bacterial cell and/or a plant cell, in vivo, in vitro or ex vivo (e.g., any cell discussed herein wherein the cell comprises an isolated cell). In an embodiment this disclosure or the inventions herein provide uses of the gene editing system embodiments and/or the compositions disclosed herein, such as in any of the foregoing paragraphs; for instance, use in treating or addressing a genetic condition of a subject,

In an embodiment, this disclosure or the inventions herein provide methods of genetically modifying a cell comprising: contacting a gene editing system as herein discussed, such as in any of the foregoing paragraphs, or a composition as herein discussed, such as in any of the foregoing paragraphs (which comprises a gene editing system as herein discussed, such as in any of the foregoing paragraphs), advantageously a gene editing system that includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, said method comprising contacting the composition or the gene editing system with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide RNA, wherein said guide RNA directs the complex to the target sequence; the nucleic acid programmable nuclease creates a double-stranded break in in the target sequence; the retron reverse transcriptase and engineered retron ncRNA create RT DNA that comprises the donor polynucleotide; and the donor polynucleotide becomes integrated at the target sequence; whereby editing the cell is genetically modified. In an embodiment, the cell can be a eukaryotic or a prokaryotic cell or an animal cell or a mammalian cell or a human cell or a bacterial cell or a plant cell.

Additional exemplary and non-limiting aspects and embodiments of the disclosure are summarized as follows in the form of numbered paragraphs.

1. An engineered nucleic acid construct comprising:

-   -   a) a first polynucleotide encoding a non-coding RNA (ncRNA),         said first polynucleotide comprising:         -   1) an msr locus encoding the msr RNA portion of a multi-copy             single-stranded DNA (msDNA); and         -   2) an msd locus encoding the msd RNA portion of the msDNA;             and     -   b) one or more heterologous nucleic acids inserted at or within         a location selected from: the msd locus, upstream of the msr         locus, upstream of the msd locus, and downstream of the msd         locus,     -   wherein the ncRNA comprises:     -   (I) an ncRNA listed in Table B, or an ncRNA having at least 50%,         at least 55%, at least 60%, at least 65%, at least 70%, at least         75%, at least 80%, at least 85%, at least 90%, at least 91%, at         least 92%, at least 93%, at least 94%, at least 95%, at least         96%, at least 97%, at least 98%, at least 99%, at least 99.1%,         at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,         at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%         sequence identity with an ncRNA listed in Table B; and/or     -   (II) an ncRNA having a conserved structure of any one of the         ncRNA structures of FIGS. 2-27 ; and     -   wherein the ncRNA optionally excludes any ncRNA associated in         nature with any one of the retron reverse transcriptases of         Table X.         2. The engineered nucleic acid construct of paragraph 1, further         comprising a second polynucleotide encoding a reverse         transcriptase (RT), or a portion thereof, wherein the encoded RT         or portion thereof is capable of synthesizing a DNA copy of at         least a portion of the msd locus encoding the msDNA.         3. The engineered nucleic acid construct of paragraph 2,     -   wherein the second polynucleotide comprises:     -   III) a polynucleotide listed in Table A, or a polynucleotide         having at least 50%, at least 55%, at least 60%, at least 65%,         at least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 91%, at least 92%, at least 93%, at least 94%, at         least 95%, at least 96%, at least 97%, at least 98%, at least         99%, at least 99.1%, at least 99.2%, at least 99.3%, at least         99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least         99.8% or at least 99.9% sequence identity to a polynucleotide         listed in Table A; and/or     -   IV) encodes a consensus amino acid sequence of Table C, or         encodes an amino acid sequence having at least 50%, at least         55%, at least 60%, at least 65%, at least 70%, at least 75%, at         least 80%, at least 85%, at least 90%, at least 91%, at least         92%, at least 93%, at least 94%, at least 95%, at least 96%, at         least 97%, at least 98%, at least 99%, at least 99.1%, at least         99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least         99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence         identity to an amino acid sequence listed in Table C; and/or     -   wherein the second polynucleotide encodes:     -   V) a polypeptide listed in Table A, or a polypeptide having at         least 50%, at least 55%, at least 60%, at least 65%, at least         70%, at least 75%, at least 80%, at least 85%, at least 90%, at         least 91%, at least 92%, at least 93%, at least 94%, at least         95%, at least 96%, at least 97%, at least 98%, at least 99%, at         least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at         least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or         at least 99.9% sequence identity to a polypeptide listed in         Table A; and/or     -   VI) a polypeptide comprising a polypeptide consensus sequence         listed in Table C, or a polypeptide having at least 50%, at         least 55%, at least 60%, at least 65%, at least 70%, at least         75%, at least 80%, at least 85%, at least 90%, at least 91%, at         least 92%, at least 93%, at least 94%, at least 95%, at least         96%, at least 97%, at least 98%, at least 99%, at least 99.1%,         at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,         at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%         sequence identity to an amino acid sequence listed in Table C;         and/or     -   wherein the second polynucleotide optionally does not encode an         amino acid sequence listed in Table X.         4. An engineered nucleic acid construct comprising:     -   a) a first polynucleotide encoding a non-coding RNA (ncRNA),         said first polynucleotide comprising:         -   1) an msr locus encoding the msr RNA portion of a multi-copy             single-stranded DNA (msDNA); and         -   2) an msd locus encoding the msd RNA portion of the msDNA;     -   b) one or more heterologous nucleic acids inserted at or within         a location selected from: the msd locus, upstream of the msr         locus, upstream of the msd locus, and downstream of the msd         locus; and     -   c) a second polynucleotide encoding a reverse transcriptase         (RT), or a portion thereof, wherein the encoded RT or portion         thereof is capable of synthesizing a DNA copy of at least a         portion of the msd locus encoding the msDNA, and,     -   wherein the non-coding RNA (ncRNA) of the first polynucleotide         optionally has a conserved structure of any one of the ncRNA         structures of FIGS. 2-27 ;     -   wherein the second polynucleotide comprises:     -   I) a polynucleotide listed in Table A, or a polynucleotide         having at least 50%, at least 55%, at least 60%, at least 65%,         at least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 91%, at least 92%, at least 93%, at least 94%, at         least 95%, at least 96%, at least 97%, at least 98%, at least         99%, at least 99.1%, at least 99.2%, at least 99.3%, at least         99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least         99.8% or at least 99.9% sequence identity to a polynucleotide         listed in Table A; and/or     -   wherein the second polynucleotide encodes:     -   II) a polypeptide listed in Table A, or a polypeptide having at         least 50%, at least 55%, at least 60%, at least 65%, at least         70%, at least 75%, at least 80%, at least 85%, at least 90%, at         least 91%, at least 92%, at least 93%, at least 94%, at least         95%, at least 96%, at least 97%, at least 98%, at least 99%, at         least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at         least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or         at least 99.9% sequence identity to a polypeptide listed in         Table A; and/or     -   IV) a polypeptide comprising a polypeptide consensus sequence         listed in Table C, or a polypeptide having at least 50%, at         least 55%, at least 60%, at least 65%, at least 70%, at least         75%, at least 80%, at least 85%, at least 90%, at least 91%, at         least 92%, at least 93%, at least 94%, at least 95%, at least         96%, at least 97%, at least 98%, at least 99%, at least 99.1%,         at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,         at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%         sequence identity to a polypeptide listed in Table C; and     -   wherein the second polynucleotide optionally does not encode an         amino acid sequence of Table X.         4a. An engineered nucleic acid construct, comprising:     -   1) an msr locus (that encodes the msr RNA portion of an msDNA);     -   2) an msd locus encoding the msd RNA portion of the msDNA;     -   3) a sequence encoding a retron reverse transcriptase (RT),         wherein said msd RNA is capable of being reverse transcribed to         form the msDNA by the retron reverse transcriptase (RT); and,     -   4) a heterologous nucleic acid inserted at or within the msd         locus, upstream of the msr locus, upstream or downstream of the         msd locus;     -   wherein the engineered nucleic acid construct optionally has (a)         a secondary structure of a wild-type ncRNA of any one of FIGS.         2-27 or     -   b) a variant of a), having:         -   i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per             10 red lettered-nucleotides;         -   ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes             per 10 black lettered-nucleotides; and/or         -   iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide             changes per 10 grey lettered-nucleotides; and/or         -   optionally further comprising:         -   i) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10             red-circled nucleotides;         -   ii) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present             per 10 black-circled nucleotides;         -   iii) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10)             nucleotides present per 10 grey-circled nucleotides; and/or         -   iv) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9,             or 10) nucleotides present per 10 white-circled nucleotides.             5. The engineered nucleic acid construct of any one of             paragraphs 1 to 4a, comprising one or more sequence             modifications (e.g., an insertion, deletion, and/or             substitution of one or more nucleotide(s)) in the msr locus             and/or the msd locus that:     -   a) modulates (e.g., enhances) reverse transcription,         processivity, accuracy/fidelity, and/or production of the msDNA         (e.g., in the mammalian cell);     -   b) modulates (e.g., reduces) immunogenicity of ncRNA encoded by         the engineered retron (e.g., the msr locus and/or the msd locus)         in a host (e.g., a host comprising the mammalian cell);     -   c) modulates (e.g., inhibits, either permanently or transiently)         a function of the msDNA; and/or     -   d) modulates (e.g., improves) efficiency of targeted genome         editing/engineering.         6. The engineered nucleic acid construct of any one of         paragraphs 1 to 4, wherein said engineered nucleic acid         construct has a secondary structure of a wild-type retron         encoding a wild-type retron ncRNA encompassed by:     -   a) any one of the structures as depicted in FIGS. 2-27 , or     -   b) a variant of a), having:         -   i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per             10 red lettered-nucleotides;         -   ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes             per 10 black lettered-nucleotides; and/or         -   iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide             changes per 10 grey lettered-nucleotides; and/or     -   optionally further comprising:         -   i) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10             red-circled nucleotides;         -   ii) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present             per 10 black-circled nucleotides;         -   iii) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10)             nucleotides present per 10 grey-circled nucleotides; and/or         -   iv) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9,             or 10) nucleotides present per 10 white-circled nucleotides.             7. The engineered nucleic acid construct of any one of             paragraphs 1-6, wherein the nucleic acid construct is             engineered by introducing the one or more sequence             modifications into a wild-type retron encoding a wild-type             ncRNA listed in Table B.             8. The engineered nucleic acid construct of any one of             paragraphs 1-7, wherein the one or more sequence             modifications in the ncRNA comprises one or more of:     -   (i) a modified (e.g., mutated, reduced, or eliminated) bulge in         a1, a2, or both a1 and a2;     -   (ii) an extension or shortening of a1, a2, or both a1 and a2;     -   (iii) an extension or shortening of a spacer sequence between         hairpin loops (e.g., S1, S2, S3, and/or S4);     -   (iv) an additional or modified (e.g., mutated or eliminated)         bulge in hairpin loops (e.g., L2 and/or L3 (e.g., by removing         unpaired bases in the bulge, or by replacing unpaired bases with         an equivalent number of base pairs));     -   (v) a modified (e.g., extended or shortened) length of hairpin         loops (e.g., L1, L2, L3, and/or L4);     -   (vi) an alternative L1 and/or L2 having complement, reverse, or         reverse complement sequences;     -   (vii) a modified (e.g., increased) number of unpaired bases at         the tip of hairpin loops (e.g., L1, L2, L3, and/or L4);     -   (viii) a modified (e.g., increased or decreased) GC content in         hairpin loops (e.g., L1, L2, L3, and/or L4);     -   (ix) an insertion of the heterologous nucleic acid in spacer         sequences between hairpin loops (e.g., S1, S2, S3 and/or S4), or         at the tip of hairpin loops (e.g., L1, L2, L3, and/or L4);     -   (x) a deletion of one or more hairpin loops (e.g., L1, L2, L3         and/or L4);     -   (xi) an addition of a new loop in a spacer sequence between         hairpin loops (e.g., S1, S2, S3, and/or S4);     -   (xii) circularization of the ncRNA with the 5′ end and the 3′         end of the ncRNA being connected either directly, or via a         spacer sequence;     -   (xiii) a repositioned branching guanosine capable of initiating         reverse transcription priming;     -   (xiv) a staggered end sequence that reduces immunogenicity of         the retron ncRNA, created by, e.g., adding or removing the 5′ a1         nucleotides and/or the 3′ a2 nucleotides; and/or,     -   (xv) an antisense sequence complementary to a CRISPR/Cas guide         RNA (gRNA) sequence encoded by the heterologous nucleic acid,         wherein the antisense sequence hybridizes to and inhibits said         gRNA in the encoded retron ncRNA, and wherein said antisense         sequence is removed upon reverse transcription of the msDNA.         9. The engineered nucleic acid construct of any one of         paragraphs 1-8, wherein the one or more heterologous nucleic         acid sequences comprise:     -   a) a heterologous nucleic acid (such as the coding sequence for         an RNA aptamer or a ribozyme) inserted into the msr locus or the         msd locus (such as in an S region (e.g., S1, S2, S3 and/or S4),         or the tip of an L region (e.g., L1, L2, L3 and/or L4), or         upstream or downstream of either the msr locus or the msd locus;         or     -   b) a first heterologous nucleic acid inserted into the msd         locus, and a second heterologous nucleic acid inserted either         upstream of the msr locus or downstream of the msd locus,         wherein the second heterologous nucleic acid encodes a guide         RNA.         10. The engineered nucleic acid construct of any one of         paragraphs 1-9, wherein said heterologous nucleic acid encodes:

-   (a) a protein or peptide of interest, or wherein said heterologous     nucleic acid comprises;

-   (b) a DNA donor template sequence;

-   (c) a functional DNA element selected from a promoter, an enhancer,     a protein binding sequence, a methylation site, a homology region     for assisting gene editing, and the like; or

-   (d) a coding sequence for a functional RNA element selected from a     guide RNA and a ncRNA.     11. The engineered nucleic acid construct of paragraph 10, wherein     said protein or peptide of interest comprises a therapeutic protein     useful in treating a disease.     12. The engineered nucleic acid construct of paragraph 10, wherein     said DNA donor template sequence corrects/repairs/removes a mutation     at the target genome site.     13. The engineered nucleic acid construct of any one of paragraphs     1-12, further comprising or encoding a sequence-specific nuclease     (such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a     meganuclease, TnpB, IscB, or a restriction endonuclease (RE)),     and/or a DNA-repair modulating biomolecule.     13b. The engineered nucleic acid construct of paragraphs 1-13     wherein the engineered nucleic acid is an all-RNA component system.     13c. The engineered nucleic acid construct of paragraphs 1-13     wherein the engineered nucleic acid is an all-DNA molecule system.     14. The engineered nucleic acid construct of paragraph 13, wherein     the sequence-specific nuclease is fused to the RT, optionally via a     flexible linker (e.g., a flexible linker comprising Gly and Ser rich     sequences such as G4S repeats or GS repeats) or by a generally     disordered protein sequence (such as unstructured hydrophilic,     biodegradable protein polymer, e.g., an XTEN peptide polymer).     15. The engineered nucleic acid construct of paragraph 13 or 14,     wherein the nuclease is a CRISPR/Cas effector enzyme that forms a     complex with a guide RNA (gRNA) recognizing a target sequence,     wherein the gRNA is linked to the ncRNA and/or the msDNA, either     directly or through a linker/spacer polynucleotide.     16. The engineered nucleic acid construct of paragraph 13, wherein     the DNA-repair modulating biomolecule is a regulatory protein that     modulates (e.g., enhances) HDR, and the regulatory protein is fused     to the RT or to the sequence-specific nuclease, optionally via a     flexible linker (e.g., the flexible linker comprising Gly and Ser     rich sequences such as G4S repeats or GS repeats) or by a generally     disordered protein sequence (such as unstructured hydrophilic,     biodegradable protein polymer, e.g., an XTEN peptide polymer).     17. A vector system comprising one or more vectors comprising the     engineered nucleic acid construct of any one of paragraphs 1-16,     wherein the vector system is optionally all-RNA.     18. The vector system of paragraph 17, wherein the msr locus, the     msd locus, and the polynucleotide encoding the RT are comprised     within the same vector.     19. The vector system of paragraph 17 or 18, wherein the same vector     further comprises a promoter operably linked to the msr locus and/or     the msd locus.     20. The vector system of paragraph 19, wherein the promoter is     further operably linked to the polynucleotide encoding the RT.     21. A vector system comprising one or more vectors, comprising the     engineered nucleic acid construct of paragraph 1 or 2, wherein the     vector system further comprises a second polynucleotide encoding a     reverse transcriptase (RT), or a portion thereof, wherein the     encoded RT is capable of synthesizing a DNA copy of at least a     portion of the msd locus encoding the msDNA, and wherein the msr     locus, the msd locus, and the second polynucleotide encoding the RT     are provided by at least two different vectors.     22. The vector system of paragraph 21, wherein:     -   a) the second polynucleotide comprises:         -   i) a polynucleotide listed in Table A, or a polynucleotide             having at least 50%, at least 55%, at least 60%, at least             65%, at least 70%, at least 75%, at least 80%, at least 85%,             at least 90%, at least 91%, at least 92%, at least 93%, at             least 94%, at least 95%, at least 96%, at least 97%, at             least 98%, at least 99%, at least 99.1%, at least 99.2%, at             least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,             at least 99.7%, at least 99.8% or at least 99.9% sequence             identity to a polynucleotide listed in Table A; and/or     -   b) the second polynucleotide encodes:         -   i) a polypeptide listed in Table A, or a polypeptide having             at least 50%, at least 55%, at least 60%, at least 65%, at             least 70%, at least 75%, at least 80%, at least 85%, at             least 90%, at least 91%, at least 92%, at least 93%, at             least 94%, at least 95%, at least 96%, at least 97%, at             least 98%, at least 99%, at least 99.1%, at least 99.2%, at             least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,             at least 99.7%, at least 99.8% or at least 99.9% sequence             identity to a polypeptide listed in Table A; and/or         -   ii) a polypeptide listed in Table C, or a polypeptide having             at least 50%, at least 55%, at least 60%, at least 65%, at             least 70%, at least 75%, at least 80%, at least 85%, at             least 90%, at least 91%, at least 92%, at least 93%, at             least 94%, at least 95%, at least 96%, at least 97%, at             least 98%, at least 99%, at least 99.1%, at least 99.2%, at             least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,             at least 99.7%, at least 99.8% or at least 99.9% sequence             identity to a polypeptide listed in Table C; and     -   wherein the second polynucleotide optionally does not encode a         polypeptide listed in Table X.         23. The vector system of paragraph 21 or 22, wherein the         polynucleotide encoding the RT is provided in trans with respect         to the msr gene and/or the msd gene.         24. The vector system of any one of paragraphs 17-23, wherein         the one or more vectors comprise a viral vector.         25. The vector system of paragraph 24, wherein the viral vector         is a retroviral vector, a lentiviral vector, an adenoviral         vector, an adeno-associated viral vector, a vaccinia viral         vector, a poxviral vector, or a herpes simplex viral vector.         26. The vector system of any one of paragraphs 17-23, wherein         the one or more vectors comprise a non-viral vector.         27. The vector system of paragraph 26, wherein the non-viral         vector comprises a plasmid.         28. The vector system of paragraph 26, wherein the non-viral         vector comprises a liposome, a lipid nanoparticle (LNP), a         cationic polymer, a vesicle, or a gold nanoparticle.         29. The vector system of any one of paragraphs 17-28, comprising         a vector encoding a sequence-specific nuclease.         30. The vector system of paragraph 29, wherein the         sequence-specific nuclease comprises an RNA-guided         sequence-specific nuclease (e.g., a CRISPR/Cas effector enzyme,         an engineered RNA-guided FokI-nuclease (e.g., dCas-FokI), an         RNA-guided DNA endonuclease, TnpB, IscB, or a         transposon-associated nuclease), or a non-RNA-guided         sequence-specific nuclease (e.g., a meganuclease, a zinc finger         nuclease (ZFN), a TALE nuclease (TALEN), or a restriction         endonuclease (RE)).         31. The vector system of paragraph 30, wherein the Cas effector         enzyme is a Class 1, Type I, II, or III Cas; a Class 2, Type II         Cas (e.g., Cas9); or a Class 2, Type V Cas (e.g., Cpf1).         32. The vector system of paragraph 30, wherein:     -   1) the RNA-guided sequence-specific nuclease comprises the         CRISPR/Cas effector enzyme, the engineered RNA-guided         FokI-nuclease (e.g., dCas-FokI), the RNA-guided DNA         endonuclease, TnpB, IscB, IsrB, or the transposon-associated         nuclease; or,     -   2) non-RNA-guided sequence-specific nuclease comprises the         meganuclease, the zinc finger nuclease (ZFN), the TALE nuclease         (TALEN), or the restriction endonuclease (RE).         33. The vector system of any one of paragraphs 17-32, further         comprising a vector encoding a homologous recombination enhancer         protein.         34. An RNA molecule encoded by the engineered nucleic acid         construct of any one of paragraphs 1-16.         35. An engineered nucleic acid-enzyme construct comprising:     -   a) a non-coding RNA (ncRNA) comprising:         -   i) an msr locus encoding the msr RNA portion of a multi-copy             single-stranded DNA (msDNA); and         -   ii) an msd locus encoding the msd RNA portion of the msDNA;     -   b) a heterologous nucleic acid inserted at or within a location         selected from: the msd locus, upstream of the msr locus,         upstream of the msd locus, and downstream of the msd locus; and     -   c) a sequence encoding a reverse transcriptase (RT), or a domain         thereof comprising:         -   i) a polypeptide listed in Table A, or a polypeptide having             at least 50%, at least 55%, at least 60%, at least 65%, at             least 70%, at least 75%, at least 80%, at least 85%, at             least 90%, at least 91%, at least 92%, at least 93%, at             least 94%, at least 95%, at least 96%, at least 97%, at             least 98%, at least 99%, at least 99.1%, at least 99.2%, at             least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,             at least 99.7%, at least 99.8% or at least 99.9% sequence             identity to a polypeptide listed in Table A; and/or         -   ii) a polypeptide listed in Table C, or a polypeptide having             at least 50%, at least 55%, at least 60%, at least 65%, at             least 70%, at least 75%, at least 80%, at least 85%, at             least 90%, at least 91%, at least 92%, at least 93%, at             least 94%, at least 95%, at least 96%, at least 97%, at             least 98%, at least 99%, at least 99.1%, at least 99.2%, at             least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%,             at least 99.7%, at least 99.8% or at least 99.9% sequence             identity to a polypeptide listed in Table C; and     -   wherein, the RT optionally does not comprise a polypeptide         listed in Table X.         36. An engineered nucleic acid-enzyme construct comprising:     -   a) a non-coding RNA (ncRNA) comprising:         -   i) an msr locus encoding the msr RNA portion of a multi-copy             single-stranded DNA (msDNA); and         -   ii) an msd locus encoding the msd RNA portion of the msDNA;     -   wherein the ncRNA comprises:         -   i) an ncRNA listed in Table B, or an ncRNA having at least             50%, at least 55%, at least 60%, at least 65%, at least 70%,             at least 75%, at least 80%, at least 85%, at least 90%, at             least 91%, at least 92%, at least 93%, at least 94%, at             least 95%, at least 96%, at least 97%, at least 98%, at             least 99%, at least 99.1%, at least 99.2%, at least 99.3%,             at least 99.4%, at least 99.5%, at least 99.6%, at least             99.7%, at least 99.8% or at least 99.9% sequence identity to             an ncRNA listed in Table B; and/or         -   ii) an ncRNA having a conserved structure of any one of the             ncRNA structures of FIGS. 2-27 ; and         -   wherein the ncRNA optionally excludes any ncRNA associated             in nature with any one of the retron reverse transcriptases             of Table X;     -   b) a heterologous nucleic acid inserted at or within a location         selected from: the msd locus; upstream of the msr locus;         upstream of the msd locus; and downstream of the msd locus; and     -   c) a reverse transcriptase (RT), or a portion thereof, wherein         the RT is capable of synthesizing a DNA copy of at least a         portion of the msd locus encoding the msDNA.         37. An engineered nucleic acid-enzyme construct comprising:     -   a) a non-coding RNA (ncRNA) comprising:         -   i) an msr locus encoding the msr RNA portion of a multi-copy             single-stranded DNA (msDNA); and         -   ii) an msd locus encoding the msd RNA portion of the msDNA;     -   wherein, the ncRNA comprises:         -   i) an ncRNA listed in Table B, or an ncRNA having at least             50%, at least 55%, at least 60%, at least 65%, at least 70%,             at least 75%, at least 80%, at least 85%, at least 90%, at             least 91%, at least 92%, at least 93%, at least 94%, at             least 95%, at least 96%, at least 97%, at least 98%, at             least 99%, at least 99.1%, at least 99.2%, at least 99.3%,             at least 99.4%, at least 99.5%, at least 99.6%, at least             99.7%, at least 99.8% or at least 99.9% sequence identity to             an ncRNA listed in Table B; and/or         -   ii) an ncRNA having a conserved structure of any one of the             ncRNA structures of FIGS. 2-27 ; and         -   wherein the ncRNA optionally excludes any ncRNA associated             in nature with any one of the retron reverse transcriptases             of Table X;     -   b) a heterologous nucleic acid inserted at or within a location         selected from: the msd locus, upstream of the msr locus,         upstream of the msd locus, and downstream of the msd locus; and     -   c) a reverse transcriptase (RT) or a domain thereof:     -   wherein the RT comprises:     -   i) an RT listed in Table A, or an RT having at least 50%, at         least 55%, at least 60%, at least 65%, at least 70%, at least         75%, at least 80%, at least 85%, at least 90%, at least 91%, at         least 92%, at least 93%, at least 94%, at least 95%, at least         96%, at least 97%, at least 98%, at least 99%, at least 99.1%,         at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%,         at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9%         sequence identity to an RT listed in Table A; and/or     -   ii) a consensus sequence listed in Table C, or a polypeptide         having at least 50%, at least 55%, at least 60%, at least 65%,         at least 70%, at least 75%, at least 80%, at least 85%, at least         90%, at least 91%, at least 92%, at least 93%, at least 94%, at         least 95%, at least 96%, at least 97%, at least 98%, at least         99%, at least 99.1%, at least 99.2%, at least 99.3%, at least         99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least         99.8% or at least 99.9% sequence identity to an amino acid         sequence listed in Table C; and     -   wherein the RT does not optionally comprise an RT listed in         Table X.         38. An isolated host cell comprising the engineered nucleic acid         construct of any one of paragraphs 1-16, the vector system of         any of paragraphs 17-33, the RNA molecule of paragraph 34, or         the engineered nucleic acid-enzyme construct of any one of         paragraphs 35-37.         39. The isolated host cell of paragraph 38, wherein the host         cell is a prokaryotic, archeon, or eukaryotic host cell.         40. The isolated host cell of paragraph 38, wherein the         eukaryotic host cell is a mammalian host cell.         41. The isolated host cell of paragraph 39, wherein the         eukaryotic host cell is a non-human host cell.         42. The isolated host cell of paragraph 40, wherein the         mammalian host cell is a human host cell.         43. The isolated host cell of any one of paragraphs 38-42,         wherein the host cell is an artificial cell or genetically         modified cell.         44. A pharmaceutical composition comprising:     -   a) the engineered nucleic acid construct of any one of         paragraphs 1-16, ncRNA encoded by the engineered nucleic acid         construct of any one of paragraphs 1-16, the vector system of         any one of paragraphs 17-33, the RNA molecule of paragraph 34,         the engineered nucleic acid-enzyme construct of any one of         paragraphs 35-37, and/or the isolated host cell of any one of         paragraphs 38-43; and     -   b) a pharmaceutically acceptable carrier.         45. A pharmaceutical composition comprising:     -   a) a lipid nanoparticle (LNP); and     -   b) the engineered nucleic acid construct of any one of         paragraphs 1-16, ncRNA encoded by the engineered nucleic acid         construct of any one of paragraphs 1-16, the vector system of         any one of paragraphs 17-33, the RNA molecule of paragraph 34,         and/or the engineered nucleic acid-enzyme construct of any one         of paragraphs 35-37.         46. The pharmaceutical composition of paragraph 45, wherein the         LNP encapsulates the engineered nucleic acid construct, ncRNA,         vector system, RNA molecule, and/or engineered nucleic         acid-enzyme construct.         47. The pharmaceutical composition of paragraph 45 or 46,         wherein the lipid nanoparticle comprises:     -   a) one or more ionizable lipids;     -   b) one or more structural lipids;     -   c) one or more PEGylated lipids; and     -   d) one or more phospholipids.         48. The pharmaceutical composition of paragraph 47, wherein the         one or more ionizable lipids is selected from the group         consisting of those disclosed in Table 2.         49. The pharmaceutical composition of paragraph 47 or 48,         wherein the one or more structural lipids are selected from the         group consisting of cholesterol, fecosterol, beta sitosterol,         sitosterol, ergosterol, campesterol, stigmasterol,         brassicasterol, tomatidine, tomatine, ursolic acid,         alpha-tocopherol, prednisolone, dexamethasone, prednisone, and         hydrocortisone.         50. The pharmaceutical composition of any one of paragraphs         47-49, wherein the one or more PEGylated lipids are selected         from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE,         PEG-DMPE, PEG-DPPC, and PEG-DSPE.         51. The pharmaceutical composition of any one of paragraphs         47-50, wherein the one or more phospholipids are selected from         the group consisting of         1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),         1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),         1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC),         1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC),         1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),         1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),         1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC),         1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocho line (POPC),         1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether         PC), 1-oleoyl-2-cholesterylhemisuc         cinoyl-sn-glycero-3-phosphocholine (OChemsPC),         1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC),         1,2-dilinolenoyl-sn-glycero-3-phosphocholine,         1,2-diarachidonoyl-sn-glycero-3-phosphocholine,         1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine,         1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE),         1,2-distearoyl-sn-glycero-3-phosphoethanolamine,         1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine,         1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine,         1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine,         1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine,         1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt         (DOPG), and sphingomyelin.         52. The pharmaceutical composition of any one of paragraphs         47-51, wherein the lipid nanoparticle comprises about 48.5 mol %         ionizable lipid, about 10 mol % phospholipid, about 40 mol %         structural lipid, and about 1.5 mol % of PEG lipid.         53. The pharmaceutical composition of any one of paragraphs         47-52, wherein the lipid nanoparticle comprises about 48.5 mol %         ionizable lipid, about 10 mol % phospholipid, about 39 mol %         structural lipid, and about 2.5 mol % of PEG lipid.         54. The pharmaceutical composition of any one of paragraphs         47-53, wherein the LNP further comprises a targeting moiety         operably connected to the LNP.         55. The pharmaceutical composition of any one of paragraphs         47-54, wherein the LNP further comprises one or more additional         components selected from the group consisting of DDAB, EPC,         14PA, 18BMP, DODAP, DOTAP, and C12-200.         56. The pharmaceutical composition of paragraph 45, wherein the         lipid nanoparticle comprises at least one cationic lipid         selected from the group consisting of: a lipid in Table 2, a         lipid having a structure of Formula (I), a lipid having a         structure of Formula (II), a lipid having a structure of Formula         (III), a lipid having a structure of Formula (IV), a lipid         having a structure of Formula (V), a lipid having a structure of         Formula (VI), and combinations thereof.         57. A kit comprising the engineered nucleic acid construct of         any one of paragraphs 1-16, ncRNA encoded by the engineered         nucleic acid construct of any one of paragraphs 1-16, the vector         system of any one of paragraphs 17-33, the RNA molecule of         paragraph 34, the engineered nucleic acid-enzyme construct of         any one of paragraphs 35-37, the host cell of any one of         paragraphs 38-43, or the pharmaceutical composition of any one         of paragraphs 44-56, and instructions for genetically modifying         a cell with said engineered nucleic acid construct, ncRNA,         vector system, host cell, or pharmaceutical composition.         58. A method of modifying a target DNA sequence in a host (e.g.,         mammalian) cell, the method comprising introducing into the         mammalian cell the engineered nucleic acid construct of any one         of paragraphs 1-16, ncRNA encoded by the engineered nucleic acid         construct of any one of paragraphs 1-16, the vector system of         any one of paragraphs 17-33, the RNA molecule of paragraph 34,         the engineered nucleic acid-enzyme construct of any one of         paragraphs 35-37, or the pharmaceutical composition of any one         of paragraphs 44-56, to allow production of the msDNA in the         host (e.g., mammalian) cell, wherein the heterologous nucleic         acid in the msDNA is integrated into the genome of the host         (e.g., mammalian) cell at the target DNA sequence by         homology-dependent recombination.         59. The method of paragraph 58, wherein the modifying comprises         introducing an insertion, deletion and/or substitution into the         target DNA sequence.         60. A method of treating a disease or condition in a subject in         need thereof, the method comprising administering a         therapeutically effective amount of the engineered nucleic acid         construct of any one of paragraphs 1-16, ncRNA encoded by the         engineered nucleic acid construct of any one of paragraphs 1-16,         the vector system of any one of paragraphs 17-33, the RNA         molecule of paragraph 34, the engineered nucleic acid-enzyme         construct of any one of paragraphs 35-37, the host cell of any         one of paragraphs 38-43, or the pharmaceutical composition of         any one of paragraphs 44-56 to the subject, thereby treating the         disease or condition in the subject.         61. A method of treating a disease or condition in a subject in         need thereof, the method comprising administering a         therapeutically effective amount of the host cell of any one of         paragraphs 38-43 to the subject, thereby treating the disease or         condition in the subject.         62. The method of paragraph 61, wherein the host cell is         autologous to the subject.         63. The method of paragraph 61, wherein the host cell is         allogeneic to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A is a schematic that depicts naturally occurring retrons from genomic DNA stage through production of the msDNA chimeric microsatellite molecule. Retrons are encoded in the bacterial genome and comprise a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT). The ncRNA and the RT initially are transcribed from the retron DNA as a single polycistronic message. The initial transcript is processed resulting in the removal or separation of the transcript encoding the retron RT. The remaining transcript is the ncRNA, which undergoes folding to form a secondary structure having several characteristic stem-loops and a duplex formed between the 5′ and 3′ regions of the ncRNA (i.e., the a1/a2 duplex). The folded ncRNA is recognized by the accompanying RT which is separately translated and provided in trans. The translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region. The RT initiates reverse transcription of the RNA towards its 5′ end, starting from the 2′-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA. A portion (i.e., the msd region) of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus. During reverse transcription, cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA. The result of the reverse transcription, the msDNA (lower right of schematic), remains covalently attached to the RNA template via the 2′-5′ phosphodiester bond, and base-pairs with the RNA template using the 3′ end of the msDNA.

FIG. 1B is a schematic depicting an embodiment of a recombinant retron contemplated by this disclosure. In this embodiment, a nucleic acid molecule comprises a nucleotide sequence encoding a retron ncRNA region (the msr/msd region) is depicted at the top left. The msr region has been modified by introducing one or more nucleotide modifications (e.g., a nucleotide substitution, deletion, or insertion). For example, it may be desirable to introduce one or more nucleotide substitutions in the msr to enhance functionality (e.g., binding of the corresponding ncRNA to RT, improved stability, improved folding, etc.). The modified msr is referred to as msr′. In addition, the msd has been modified by introducing a heterologous nucleotide sequence encoding an HDR donor template. Lastly, the retron DNA has been modified to introduce a nucleotide sequence encoding a guide RNA at the 3′ end of the retron DNA sequence. is configured on a DNA vector (e.g., a plasmid). The DNA is shown to be transcribed as a polycistronic message that includes the msr′/msd′ region (forming the ncRNA) which is fused at its 3′ end to the guide RNA (in other embodiments, the guide RNA could be fused to the 5′ end of the retron ncRNA. This intermediate is shown to form a complex with a reverse transcriptase provided in trans (e.g., by way of a separate expression vector or delivered mRNA). The top right schematic shows the formation of a complex between the recombinant ncRNA and the RT and the beginning of reverse transcription from the covalently-linked conserved guanosine (G) (i.e., the “priming G” or “priming guanosine”) using the msd RNA as a template sequence. Following the completion of reverse transcription and RNaseH degradation of the template RNA sequence, a recombinant msDNA is formed which comprises three modifications, as shown: (a) a guide RNA linked to the 3′ end of the msDNA, (b) a nucleotide change in the msr′, and (c) the reverse-transcribed single-strand DNA comprises a region that is an HDR donor template. Such a recombinant msDNA could then facilitate various genome modification applications in the cell, including genome editing with an RNA-guided nuclease provided to the cell in trans.

FIG. 1C is a schematic depicting a recombinant retron-based genome editing system described herein. In the case of genome editing involving an RNA-guided nuclease, the components of such a system may include (a) a guide RNA provided in cis (e.g., fused to the recombinant retron msDNA) and/or in trans (e.g., separately expressed in a cell), (b) a recombinant ncRNA (including at least a sequence encoding an HDR donor template and optionally a guide RNA fused to the ncRNA), (c) a reverse transcriptase, and (d) a programmable nuclease. These components are provided to a cell in the form of DNA and/or RNA and/or protein by a delivery means (e.g., LNPs, liposomes, virus-based delivery, or passive/active transport). Once inside the cell, the recombinant msDNA is formed. The msDNA and the programmable nuclease translocate to the nuclease to conduct gene editing at a target DNA site, thereby producing an edited DNA target.

FIG. 1D provides a simplified schematic of a the natural lifecycle of a retron. Retrons typically comprise a reverse transcriptase (RT) and two non-coding contiguous inverted sequences (msr and msd) transcribed as a single RNA that is folded into a specific secondary structure. The conserved NAXXH motif and VTG triplet in retron RTs are indicated. The RT binds downstream from the msd locus in the RNA, initiating reverse transcription of the RNA template towards its 5′ end, assisted by the 2′OH group present in a conserved branching G residue acting as a primer. Reverse transcription halts before the msr region is reached, and the resulting msDNA remains covalently attached to the RNA template via a 2′-5′ phosphodiester bond and base-pairing of the 3′ ends of the molecules.

FIG. 1E provides a detailed representation of the natural biological pathway of a retron in a cell, concluding with the generation of the msDNA satellite molecule. This figure parallels FIG. 1D but more completely depicts the stages of msDNA production. (1) depicts the retron locus which includes an ncRNA locus having an msr locus and a msd locus (both of which are non-coding) and a reverse transcriptase (RT) locus. The ncDNA locus and the RT locus are transcribed as a single RNA transcript, which is depicted in (2). The colors representing each component in (1) are carried through each of stages (2) through (6). Stages (3) and (4) depict the folding of the ncRNA portion into a series of stem loops, wherein the 5′ end and the 3′ ends of the ncRNA form a duplex. In addition, the position of the conserved branching guanosine residue having a 2′OH group is show. The branching guanosine serves as a future priming site for the reverse transcriptase. Stage (4) further shows that the region of the transcript encoding the reverse transcriptase is removed, separately being translated to produce the reverse transcript as enzyme. In stage (5), the reverse transcriptase associates with the folded ncRNA and begins polymerization of a single strand of DNA (i.e., the reverse transcription product) from the primer site (i.e., the conserved branching guanosine residue having the 2′OH end) and using the msd RNA sequence as a template. Reverse transcription terminates at the msr region. The msd RNA template is exonucleotically removed, thereby resulting in a chimeric molecule comprising the msr RNA region which is covalently joined to the ssDNA transcription product through covalent linkage to the conserved guanosine primer residue. There is also a short duplex region that forms between the 3′ end of the msr RNA and the 3′ of the ssDNA reverse transcript product. The complete molecule is referred to as the “msDNA.”

FIG. 1F is a schematic depicting that the herein disclosed recombinant retron-based genome modification systems may be implemented as (a) cell recorder systems, (b) genome editing systems, and (c) recombineering systems. These uses are not intended to be limiting.

FIG. 1G is a schematic depicting various configurations contemplated for the recombinant retron disclosed herein. (1) shows the operonic structure of a wild type retron; (2) shows the operonic structure of a recombinant retron configured to encode an HDR donor template in the final msDNA molecule; (3) shows (2) but further modified to encode a guide RNA at the 3′ end of the retron; (4) shows (2) but further modified to encode a guide RNA in trans.

FIG. 1H is a schematic that emphasizes that any suitable configuration for presenting the components of the recombinant retron-based genome modification systems disclosed herein to a cell are contemplated, including where the RT and/or the programmable nuclease are provided in trans relative to the retron ncRNA. In some embodiments, the RT and the programmable nuclease may be provided as a fusion protein.

FIG. 1I depicts that the RT and programmable nuclease may be provided as fusion proteins (top, middle) or provided separate from one another.

FIG. 1J depicts that nuclear localization signals may be engineered into the polypeptides of the disclosure (e.g., a RNA-guide nuclease) to facilitate translocation into the nuclease of cell where editing occurs.

FIG. 1K is a schematic (not to scale) representation depicting an embodiment of genome editing, in which double-stranded break (DSB) created by a suitable nuclease (such as a CRISPR/Cas effector enzyme, a ZFN, TALEN, meganuclease, TnpB, IscB, or restriction enzymes (Res)) promotes the insertion of a donor or template sequence (shown here as a “marker” flanked by homologous sequences matching those flanking the DSB, provided on a “donor vector”).

FIG. 2 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IA/IIA1 retron produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepairs.

FIG. 3 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IB1 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 4 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IB2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 5 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IC retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 6 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type HA other retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 7 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 8 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA3 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 9 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA4 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 10 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA5 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 11 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIA1 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 12 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIA2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 13 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of IIIA3 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 14 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIA4 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 15 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIA5 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 16 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IIIunk retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 17 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IV retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 18 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type IX retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 19 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type V retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 20 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type VI retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 21 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XI Group 1 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 22 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XI Group 2 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 23 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XII retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 24 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XIII retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 25 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Type XIV retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 26 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Ec107 retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 27 is a schematic representation of a consensus secondary structure of a retron ncRNA msr/msd of Outgroup A retrons produced by computational structural alignment of ncRNA sequences from Table B as described in Example 3.

The colored dots represents the probability a base is at that location (e.g., red circle represents the presence of a base in 97% of the cases, black represents the presence of a base in 90-97% of the cases, grey represents the presence of a base in 75-90% of the cases, and white represents the presence of a base in 50-75% of the cases), as opposed to a gap (no base), whereas the colored letters represent bases that are conserved to different degrees (e.g., with red representing 97%+ conserved, black being 90%+ conserved, and grey at least 75% conserved). Each highlighted base-pair represents a significantly covarying basepair.

FIG. 28 is a phylogenetic tree of RT sequences constructed in accordance with Example 3.

FIG. 29 is a structural representation of the retron loci associated with each retron type in FIG. 28 .

FIG. 30 shows the position of certain retrons (EcoI, Eco3, Eco5, AcoI, RTX003_2042, RTX003_6083v1, and RTX003_6943) within the phylogenetic retron tree of FIG. 28 .

FIG. 31A is a plasmid map of an exemplary retron (EcoI) tested in the Examples herein.

FIG. 31B is a linear representation in 5′ to 3′ direction of the plasmid map of FIG. 31A.

FIG. 31C is a plasmid map of an exemplary retron (RTX3_6083v1) tested in the Examples herein.

FIG. 31D is a linear representation in 5′ to 3′ direction of the plasmid map of FIG. 31C.

FIG. 32 is a representation of a plasmid-based assay used to measure retron precise edits and indels as performed in the Examples. In Step 1, a plasmid (e.g., that of FIG. 31A or FIG. 31C) is transfected into human cells (e.g., HEK293t cells) which are engineered to express Cas9. Editing is allowed to occur for 72 hours at 37° C. In Step 2, the genomic DNA is extracted from the cells and used to prepare a next-generation sequencing (NGS) library for sequencing. The library is sequenced over the target site (e.g., EMX1) of editing to generate sequence reads. In Step 3, the sequencing reads are analyzed to obtain a frequency of sequence reads containing the desired edit (percentage of precision editing) and a frequency of indels at the desired edit site (percentage of indels).

FIG. 33 is an equivalent representation of FIG. 32 .

FIG. 34 is a representation of a methodology for transfecting HEK293T cells. Cells were seeded in 24 well plate one day prior to transfection. Appropriate amount of plasmid and transfection reagent (e.g., Lipofectamine 3000) were mixed and transferred to cells. After 72 hours incubation, genomic DNA was extracted and the target edit region was amplified into sequencing libraries. Sequencing data was analyzed by CRISPResso2 and percentage of precise edit and indels were calculated.

FIG. 35 is an example of reference sequence and desired editing outcome for Eco3 retron at he EMX1 genomic site. Analysis of editing outcomes is performed using CRISPresso2 pipeline. In this example, the editing template inserts a 10 bp insertion into the FMXI gene (TTACGTCTGC) along with a 6 bp substitution to mutate the PAM sequence (GAAGGG>AAAGTT).

FIG. 36 shows the results of plasmid-based assay (e.g., according to FIG. 33 ) demonstrating up to about 0.3% precise edits and as low as 40% indels with Eco1 retron in Cas9 expressing HEK293T cells. The plasmid that encodes Eco1 RT and Eco1 ncRNA-sgRNA fusion targeting EMX1 was transfected via lipofection using two different amounts of Lipofectamine.

FIG. 37 shows the results of plasmid-based assay (e.g., according to FIG. 33 ) demonstrating up to about 0.1% precise edits and as low as 3% indels with AcoI. Aco1 retron has not been experimentally validated to produce msDNA. Precise editing activity observed in this experiment strongly support that Aco1 retron is capable of generating msDNA inside human cells.

FIG. 38 shows the results of plasmid-based assay (e.g., according to FIG. 33 ) demonstrating up to about 0.3% precise edits and as low as 5% indels with RTX003_2042. This retron can achieve a comparable precise editing to Eco1 but with significantly lower indels (10-fold). RTX003_2042 is a novel retron and precise editing activity observed in this experiment strongly support that RTX003_2042 retron could generate msDNA inside human cells.

FIG. 39A shows the results of plasmid-based assay demonstrating up to about 0.05-0.08% precise edits and as low as 2.5-4% indels with RTX003_6083vl and 6943. Both are novel retrons and precise editing activity observed in this experiment strongly support that RTX003_6083v1 and 6943 retron could generate msDNA inside human cells.

FIG. 39B shows follow up experiments using the same assay of FIG. 39A indicated that RTX3_6083v1 and RTX3_6943 generated 3-4 fold more precise edits than Eco1 while indels generated from these two retrons were 2-3 fold lower. RTX3_2042 showed precise editing at similar frequencies to Eco1 but had more variability than other samples.

FIG. 39C shows follow up experiments using the same assay of FIG. 39A indicated that RTX3_6083v1 and RTX3_6943 generated 3-4 fold more precise edits than Eco1 while indels generated from these two retrons were 2-3 fold lower. RTX3_2042 showed precise editing at similar frequencies to Eco1 but had more variability than other samples.

FIG. 40 is a representation of an two-RNA editing assay used in the Examples to measure the relative editing efficiency of exemplary retrons using electroporation-based delivery of two RNA components into HEK293T cells. Appropriate amount of RT mRNA and ncRNA-sgRNA fusion were mixed and electroporated to cells. After 72 hours incubation, genomic DNA was extracted and the targeting region was amplified into sequencing libraries. Sequencing data was analyzed by CRISPResso2 and precise edit and indels were calculated.

FIG. 41 shows the results of two-RNA system (RT mRNA+ncRNA-sgRNA fusion) delivered to Cas9 expressing HEK293T cells by electroporation. Eco1, Eco3 and Eco5 retrons were tested. Results showed precise edits (left graph) up to 0.4% for Eco3 and as low as 10% indels (right graph) for Eco3. Precise edits mediated by Eco3 increased with augmenting amount of ncRNA-sgRNA fusion from 1:2 to 1:4 ratio between RT mRNA and ncRNA-sgRNA fusion.

FIG. 42 shows the results of titration of two-component Eco3 RNA system (RT mRNA+ncRNA-sgRNA fusion) delivered to Cas9 expressing 293T cells by electroporation. The RT mRNA and the ncRNA were mixed at ratios of 1:2, 1:3, 1:4, 1:5, 1:8, 1:10, respectively, and delivered at two different amounts of RT mRNA (0.2 or 0.5 μg). On left, data showed that Eco3 at 0.5 μg produced highest percentage of precise edits at a 1:3 and 1:5 ratio of RT mRNA to ncRNA. On right, data further showed that a more equivalent ratio of RT mRNA to ncRNA resulted in a trend of lower percentage of indels.

FIG. 43 is a representation of a three-RNA retron editing system which involves delivery by electroporation of three RNA components (RT mRNA, retron ncRNA-sgRNA fusion, and Cas9 mRNA) into HEK293T cells. Appropriate amount of RT mRNA, ncRNA-sgRNA fusion, and Cas9 mRNA were mixed and electroporated to cells. After 72 hours incubation, genomic DNA was extracted and targeting region was amplified into sequencing libraries. Sequencing data was analyzed by CRISPResso2 and precise edit and indels were calculated.

FIG. 44 shows the results of Cas9 mRNA titration of three-component Eco3 RNA system (RT mRNA+ncRNA-sgRNA fusion+Cas9 mRNA) delivered to 293T cells by electroporation. The RT mRNA and the ncRNA-sgRNA fusion were mixed at given amounts on the graph and the amount Cas9 mRNA was titrated. At 0.2 μg of Cas9 mRNA, up to 0.1% of precise editing was observed. While the editing efficiency is an order of magnitude lower than two RNA system, the editing occurred by specific action of Cas9 and retron, since absence of either abrogated the editing.

FIG. 45 depicts a process of lipofection using three RNA system in HEK293T cells. Cells were seeded in 96 well plate one day prior to transfection. Appropriate amount of RT mRNA, ncRNA-sgRNA fusion, Cas9 mRNA and Lipofectamine reagent were mixed and transferred to cells. After 72 hours incubation, genomic DNA was extracted and targeting region was amplified into sequencing libraries. Sequencing data was analyzed by CRISPResso2 and precise edit and indels were calculated.

FIG. 46 shows the results of three-component Aco1 RNA system (RT mRNA+ncRNA+Cas9 mRNA) delivered to HEK293T cells by lipofection. The RT mRNA, the ncRNA and the Cas9 mRNA were mixed at amounts indicated in the graph and transfected to HEK293T cells. 56 bp insertion and 6 bp deletion at EMX1 locus was scored as precise edits and ˜0.1% of cell population has undergone precise editing on the left graph. The editing was dependent on Cas9 nuclease since its absence abrogated the editing. The frequency of indels was about 1.5% on the right graph.

FIG. 47 shows the results of minimal Cas9 nuclease activity when sgRNA is fused to ncRNA of Eco3 retron. Cas9 activity was evaluated by frequency of indels. 1 μg of ncRNA-sgRNA fusion shows 20-fold lower activity than equimolar separated sgRNA alone. In parallel, activity of chemical modified vs unmodified sgRNA was compared and the former shows 6-fold higher activity than the latter at the condition described in the graph.

FIG. 48 is a representation of the all-RNA editing assay used in the Examples to measure the relative editing efficiency of the sample retrons in an all-RNA format, modified with a step of in trans guide RNA spike-in. Electroporation using three RNA system+sgRNA trans spike-in in HEK293T cells. Appropriate amount of RT mRNA, ncRNA-sgRNA fusion, Cas9 mRNA, and sgRNA were mixed and electroporated to cells. After 72 hours incubation, genomic DNA was extracted and targeting region was amplified into sequencing libraries. Sequencing data was analyzed by CRISPResso2 and precise edit and indels were calculated.

FIG. 49 shows the results of guide RNA spike-in in all RNA system (RT mRNA+ncRNA-sgRNA fusion+Cas9 mRNA+sgRNA) delivered to HEK293T cells by electroporation. At given amount of Cas9 and RT mRNA on the graph, the amount of guide RNA spike-in is titrated at 50, 100 and 200 ng. The titration was done at two different ratios of RT mRNA: ncRNA-sgRNA fusion=1:6 or 1:8. The guide RNA spike-in in all RNA system increased precise editing up to ˜50 fold. The increasing amount of guide RNA gradually increased precise editing and 1:8 of RT mRNA:ncRNA-sgRNA fusion performed slightly better than 1:6, reaching 13% of precise editing. On the right graph, frequency of indels is shown for respective conditions.

FIG. 50 represents a lipofection process using three RNA system+gRNA trans spike-in in HEK293T cells. Cells were seeded in 96 well plate one day prior to transfection. Appropriate amount of RT mRNA, ncRNA-sgRNA fusion, Cas9 mRNA, sgRNA and Lipofectamine reagent were mixed and transferred to cells. After 72 hours incubation, genomic DNA was extracted and targeting region was amplified into sequencing libraries. Sequencing data was analyzed by CRISPResso2 and precise edit and indels were calculated.

FIG. 51 shows the results of guide RNA spike-in (i.e., delivering a separate molecule bolus of guide RNA which in all RNA system (RT mRNA+ncRNA-sgRNA fusion+Cas9 mRNA+sgRNA) delivered to HEK293T cells by lipofection. At given amount of RT mRNA, ncRNA-sgRNA fusion, and Cas9 mRNA on the graph, the amount of guide RNA spike-in is titrated at 2, 5 and 10 ng. The guide RNA spike-in in all RNA system increased precise editing up to 3.5 fold, 12% of efficiency. The increasing amount of guide RNA at this range did not further increase precise editing. The precise editing is completely dependent on the presence of Retron machinery. On the right graph, frequency of indels is shown for respective conditions.

FIG. 52 shows the results of ncRNA-sgRNA fusion separation in all RNA system (RT mRNA+Cas9 mRNA+either ncRNA-sgRNA fusion OR separate ncRNA+sgRNA) delivered to HEK293T cells by lipofection. At given amount of RT mRNA and Cas9 mRNA on the graph, the amount of guide RNA spike-in is titrated at 0, 2, 5, 10, 50 and 100 ng. At 10 ng guide RNA, precise editing peaked at 2.23% compared to 1.78% with ncRNA-sgRNA fusion. The increasing amount of guide RNA at this range did not further increase precise editing. On the right graph, frequency of indels is shown for respective conditions.

FIG. 53 is a schematic of improved templates used for in vitro transcription to produce ncRNA (left or A) and ncRNA modifications (right or B). (A) relates to the optimization of RNA production by in vitro transcription. Previously made in vitro transcription experiments to produce RNA used a double-stranded DNA template containing a 3′ overhang (on same strand as T7 promoter sequence). A new template with a blunt end was designed and tested and as shown in FIG. 54 results in increased precise editing efficiency. (B) relates to modified ncRNAs which are modified by addition of an MS2 stem loop hairpin at the 3′ end of the ncRNA. Without being bound by theory, the MS2 loop helps stabilize the ncRNA and results in significantly improved precise editing efficiency, as shown in FIG. 54 .

FIG. 54 shows the results of ncRNA-sgRNA fusion separation in 4 component all-RNA system (RT mRNA+Cas9 mRNA+ncRNA+sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount as shown on the graphs. Using RNA generated from linearized plasmid template containing a 3′ overhang produced 1.35% precise edits. Using RNA generated from the improved linearized plasmid template containing a blunt end increased precise editing to 5.94%. Adding an MS2 stem loop to the 3′ end of the ncRNA (blunt end) further increased precise editing to 12.39%. On the right graph, frequency of indels is shown for respective conditions.

FIG. 55 provides schematics for end protection of RNA from cellular nuclease activity by capping and tailing. In (A), a 7-methylguanosine cap0 was added to 5′ triphosphate of RNA. In (B), a poly-A tail was added to the 3′ end by enzymatic addition. Tail length is estimated over 50 nucleotides. In (C), RNA containing both a 5′ cap and a 3′ tail is shown. Results are shown in FIG. 56 .

FIG. 56 shows the result of end protection of ncRNA-sgRNA fusion by cap and tail in 4 component all-RNA system (RT mRNA+Cas9 mRNA+ncRNA+sgRNA) delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. ncRNA-gRNA fusion was either capped (+cap −tail) or poly-A tailed (−cap +tail) or both capped and poly-A tailed (+cap +tail). Using RNA without end protection (−cap −tail) produced ˜4.5% precise edits and the editing was dependent on retron since the absence of RT abrogated precise editing. Using RNA with either or both protection by cap and tail produced lower precise editing (left graph) but lowered indels (right graph) than without cap and tail.

DEFINITIONS

All technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & 62/1005 Marham, The Harper Collins Dictionary of Biology (1991).

General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a ncRNA” includes a plurality of ncRNAs and reference to “the reverse transcriptase” includes reference to one or more RTs and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. For example, claims may be drafted to exclude certain RT sequences.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub combination was individually and explicitly disclosed herein.

Biologically Active

As used herein, the term “biologically active” refers to a characteristic of an agent (e.g., DNA, RNA, or protein) that has activity in a biological system (including in vitro and in vivo biological system), and particularly in a living organism, such as in a mammal, including human and non-human mammals. For instance, an agent when administered to an organism has a biological effect on that organism, is considered to be biologically active.

Bulge

As used herein, the term “bulge” refers to a small region of unpaired base(s) that interrupts a “stem” of base-paired nucleotides. The bulge may comprise one or two single-stranded or unbase-paired nucleotides joined at both ends by base-paired nucleotides of the stem. The bulge can be symmetrical (viz., the two unbase-paired single-stranded regions have the same number of nucleotides), or asymmetrical (viz., the unbase-paired single stranded region(s) have different or unequal numbers of nucleotides), or there is only one unbase-paired nucleotide on one strand. A bulge can be described as A/B (such as a “2/2 bulge,” or a “1/0 bulge”) wherein A represents the number of unpaired nucleotides on the upstream strand of the stem, and B represents the number of unpaired nucleotides on the downstream strand of the stem. An upstream strand of a bulge is more 5′ to a downstream strand of the bulge in the primary nucleotide sequence.

cDNA

As used herein, the term “cDNA” refers to a strand of DNA copied from an RNA template, e.g., by a reverse transcriptase.

Complementary

As used herein, the terms “complementary” or “substantially complementary” are meant to refer to a nucleic acid (e.g., RNA, DNA) that comprises a sequence of nucleotides that enables it to non-covalently bind, i.e., form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) [DNA, RNA]. In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): guanine (G) can also base pair with uracil (U). For example, G/U base-pairing is at least partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a guanine (G) is considered complementary to both a uracil (U) and to an adenine (A). For example, when a G/U base-pair can be made at a given nucleotide position of a dsRNA duplex of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a bulge, a loop structure or hairpin structure, etc.). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656), the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489), and the like.

DNA-Guided Nuclease

As used herein, an “DNA-guided nuclease” is a type of “programmable nuclease,” and a specific type of “nucleic acid-guided nuclease.” An example of a DNA-guided nuclease is reported in Varshney et al., DNA-guided genome editing using structure-guided endonucleases, Genome Biology, 2016, 17(1), 187, which may be used in the context of the present disclosure and is incorporated herein by reference. As used herein, the term “DNA-guided nuclease” or “DNA-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide RNA thereby forming a complex between the guide RNA and the DNA-guided nuclease. The guide RNA comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence. Thus, the DNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide RNA, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson-Crick base-pairing.

DNA Regulatory Sequences

As used herein, the terms “DNA regulatory sequences,” “control elements,” and “regulatory elements,” can be used interchangeably herein to refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence and/or regulate translation of a mRNA into an encoded polypeptide.

Donor Nucleic Acid

By a “donor nucleic acid” or “donor polynucleotide” or “donor DNA” or “HDR donor DNA” it is meant a single-stranded DNA to be inserted at a site cleaved by a programmable nuclease (e.g., a CRISPR/Cas effector protein; a TALEN; a ZFN; a meganuclease) (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor polynucleotide can contain sufficient homology to a genomic sequence at the target site, e.g. 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g., within about 200 bases or less of the target site, e.g., within about 190 bases or less of the target site, e.g., within about 180 bases or less of the target site, e.g., within about 170 bases or less of the target site, e.g., within about 160 bases or less of the target site, e.g., within about 150 bases or less of the target site, e.g., within about 140 bases or less of the target site, e.g., within about 130 bases or less of the target site, e.g., within about 120 bases or less of the target site, e.g., within about 110 bases or less of the target site, e.g., within about 100 bases or less of the target site, e.g., within about 90 bases or less of the target site, e.g., within about 80 bases or less of the target site, e.g., within about 70 bases or less of the target site, e.g., within about 60 bases or less of the target site, e.g., 50 bases or less of the target site, e.g., within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology.

Encodes

As used herein, a DNA sequence that “encodes” a particular RNA is a DNA nucleotide sequence that is transcribed into RNA. A DNA polynucleotide may encode an RNA (mRNA) that is translated into protein (and therefore the DNA and the mRNA both encode the protein), or a DNA polynucleotide may encode an RNA that is not translated into protein (e.g. tRNA, rRNA, microRNA (miRNA), a “non-coding” RNA (ncRNA), a guide RNA, etc.). In the case of retrons, the retron DNA may encode the ncRNA loci (which includes the msr and msd regions) as well as a retron RT.

Engineered Retron

As used herein, the term “engineered retron” or equivalently, “recombinant retron,” refers to a retron that does not occur in nature. In one embodiment, engineered retrons can include wildtype or naturally-occurring retrons that are modified to contain at least one modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100, or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or up to 2000 nucleotides substituted, inserted, or deleted from a starting point retron (e.g., a wildtype retron). Where more than one nucleotide of a starting point retron (e.g., a wildtype retron) is substituted, deleted, or inserted, the nucleotides may be contiguous or non-contiguous. While an engineered retron as a whole is not naturally-occurring, it may include components such as nucleotide sequences that do occur in nature. For example, an engineered retron can have nucleotide sequences from different organisms (e.g., from different bacteria species), or from completely synthetic/artificial/recombinant nucleic acid sequences. Thus, an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic/artificial/recombinant nucleotide sequence, and/or combinations of such sequences. An example of modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci. Linking guide RNA molecules to the 5′ and/or 3′ ends (i.e., linking one at the 5′ end of a ncRNA and/or one at the 3′ end of a ncRNA) also represent another modification envisioned by the recombinant retrons disclosed herein. In such embodiments, the guide RNA molecules may also be categorized or referred to more generally as types of heterologous nucleic acid sequences used to modify starting point retrons.

Exosomes

As used herein, the term “exosomes” refer to small membrane bound vesicles with an endocytic origin. Without wishing to be bound by theory, exosomes are generally released into an extracellular environment from host/progenitor cells post fusion of multivesicular bodies the cellular plasma membrane. As such, exosomes can include components of the progenitor membrane in addition to designed components (e.g. engineered retron). Exosome membranes are generally lamellar, composed of a bilayer of lipids, with an aqueous inter-nanoparticle space.

Expression Vector

As used herein, the term “expression vector” or “expression construct” refers to a vector that includes one or more expression control sequences, and an “expression control sequence” is a DNA sequence that controls and regulates the transcription and/or translation of another DNA sequence. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, tobacco mosaic virus, herpes viruses, cytomegalovirus, retroviruses, vaccinia viruses, adenoviruses, and adeno-associated viruses. Numerous vectors and expression systems are commercially available, such as from Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life Technologies (Carlsbad, Calif.). The present invention comprehends recombinant vectors that may include viral vectors, bacterial vectors, protozoan vectors, DNA vectors, or recombinants thereof.

Heterologous Nucleic Acid Sequence

As used herein, the term “heterologous nucleic acid” refers to a genotypically distinct entity from that of the rest of the entity to which it is compared or into which it is introduced or incorporated. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (e.g., DNA or RNA) and, if expressed, can encode a heterologous polypeptide. Similarly, a cellular sequence (e.g., a gene or portion thereof) that is incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector. In some embodiments, the heterologous sequence inserted into the wild-type retron regions does not naturally insert into such regions (e.g., the engineered retron with the inserted heterologous sequence is not naturally existing). For example, the heterologous sequence can be from the same species of bacteria in which the wild-type retron is normally found, so long as the heterologous sequence is not naturally inserted in the wild-type retron at the location in which the heterologous sequence is inserted. In certain embodiments, the heterologous sequence is a mammalian sequence (e.g., a human sequence), or a reverse complement thereof. Heterologous nucleic acid sequences introduced into retrons can including without limitation guide RNA sequences, HDR donor templates, protein-encoding genes, or non-coding functional RNA elements (e.g., stem-loops, hairpins, and bulges).

Lipid Nanoparticle (LNP)

As used herein, the term “lipid nanoparticle” or LNP refers to a type of lipid particle delivery system formed of small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, LNPs may comprise a nucleic acid (e.g. engineered retron) into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof. In some embodiments, an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid. In alternative embodiments, an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid.

Further discuss of liposomes can be found, for example, in Tenchov et al., “Lipid Nanoparticles—From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement,” ACS Nano, 2021, 15, pp. 16982-17015 (the contents of which are incorporated by reference).

Linker

As used herein, the term“linker” refers to a molecule linking or joining two other molecules or moieties. The linker can be an amino acid sequence in the case of a linker joining two fusion proteins. For example, an RNA-guided nuclease (e.g., Cas12a) can be fused to a retron reverse transcriptase by an amino acid linker sequence. The linker can also be a nucleotide sequence in the case of joining two nucleotide sequences together. For example, in the instant case, a ncRNA at its 5′ and/or 3′ ends may be linked by a nucleotide sequence linker to one or more guide RNAs. In other embodiments, the linker is an organic molecule, group, polymer, or chemical moiety. In some embodiments, the linker is 5-100 amino acids in length, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30-35, 35-40, 40-45, 45-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-150, or 150-200 amino acids in length. Longer or shorter linkers are also contemplated.

Liposomes

As used herein, the term “liposomes” refer to a type of lipid particle delivery system comprising small vesicles that contain at least one lipid membrane surrounding an aqueous inner-nanoparticle space that is generally not derived from a progenitor/host cell. Liposomes are a versatile carrier platform in that they are capable of transporting hydrophobic or hydrophilic molecules, including small molecules, proteins, and nucleic acids into cells. They were the earliest developed generation of nanoscale medicine delivery platform. Numerous liposomal drug formulations have been approved for human medicines, e.g., Doxil, a lipid nanoparticle formulation of the antitumor agent doxorubicin. Further discuss of liposomes can be found, for example, in Tenchov et al., “Lipid Nanoparticles—From Liposomes to mRNA Vaccine Delivery, a Landscape of Diversity and Advancement,” ACS Nano, 2021, 15, pp. 16982-17015 (the contents of which are incorporated by reference).

Loop

-   -   As used herein, the term “loop” in a polynucleotide refers to a         single stranded stretch of one or more nucleotides, such as 2,         3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, wherein the most 5′         nucleotide and the most 3′ nucleotide of the loop are each         linked to a base-paired nucleotide in a stem.

Micelles

As used herein, the term “micelles” refer to small particles which do not have an aqueous intra-particle space.

Nanoparticle

As used herein, the term “nanoparticle” refers to any nanoscale particle typically ranging in size from about 1 nm to 1000 nm.

Nuclear Localization Sequence (NLS)

As used herein, the term“nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein (e.g., a RNA-guided nuclease) into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art. For example, NLS sequences are described in Plank et al., international PCT application, PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference for its disclosure of exemplary nuclear localization sequences.

Nucleic Acid

As used herein, the term “nucleic acid” or “nucleic acid molecule” or “nucleic acid sequence” or “polynucleotide” generally refer to deoxyribonucleic or ribonucleic oligonucleotides in either single- or double-stranded form. The term may also encompass oligonucleotides containing known analogues of natural nucleotides. The term also may also encompass nucleic acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. The term encompasses both ribonucleic acid (RNA) and DNA, including cDNA (including RT DNA), genomic DNA, synthetic, synthesized (e.g., chemically synthesized) DNA, and/or DNA (or RNA) containing nucleic acid analogs. The nucleotides Adenine (A), Thymine (T), Guanine (G) and Cytosine (C) also may (or may not) encompass nucleotide modifications, e.g., methylated and/or hydroxylated nucleotides, e.g., Cytosine (C) encompasses 5-methylcytosine and 5-hydroxymethylcytosine.

Nucleic Acid-Guided Nuclease

As used herein, the term “nucleic acid-guided nuclease” or “nucleic acid-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide nucleic acid (e.g., a guide RNA or a guide DNA) thereby forming a complex between the guide nucleic acid and the nucleic acid-guided nuclease. The guide nucleic acid comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence. Thus, the nucleic acid-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide nucleic acid, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson-Crick base-pairing. In some embodiments, the nucleic acid-guided nuclease will include a DNA-binding activity (e.g., as in the case for CRISPR Cas9). Most commonly, the nucleic acid-guided nuclease is programmed by associating with a guide RNA molecule and in such cases the nuclease may be called “RNA-guided nuclease.” When programmed by a guide DNA, the nuclease may be called a “DNA-guided nuclease.” Nucleic acid-guided, RNA-guided, or DNA-guided nucleases may also be referred to as “programmable nucleases,” which also include other classes of programmable nucleases which associate with specific DNA sequences through amino acid/nucleotide sequence recognition (e.g., zinc fingers nucleases (ZFN) and transcription activator like effector nucleases (TALEN)) rather than through guide RNAs. In addition, any nuclease contemplated herein may also be engineered to remove, inactivate, or otherwise eliminate one or more nuclease activities (e.g., by introducing a nuclease-inactiving mutation in the active site(s) of a nuclease). A nuclease that has been modified to remove, inactivate, or otherwise eliminate all nuclease activity may be referred to as a “dead” nuclease. A dead nuclease is not able to cut either strand of a double-stranded DNA molecule. A nuclease that has been modified to remove, inactivate, or otherwise eliminate at least one nuclease activity but which still retains at least one nuclease activity may be referred to as a “nickase” nuclease. A nickase nuclease cuts one strand of a double-stranded DNA molecule, but not both strands. For example, a CRISPR Cas9 naturally comprises two distinct nuclease activity domains, namely, the HNH domain and the RuvC domain. The HNH domain cuts the strand of DNA bound to the guide RNA and the RuvC domain cuts the protospacer strand. One can obtain a nickase Cas9 by inactivating either the HNH domain or the RuvC domain. One can obtain a dead Cas9 by inactivating both the HNH domain and the RuvC domain. Other RNA-guided nuclease may be similarly converted to nickases and/or dead nucleases by inactivating one or more of the existing nuclease domains.

Operably Linked

As used herein, the term “operably linked” or “under transcriptional control,” when used in conjunction with the description of a promoter, refers to the correct location and orientation in relation to a polynucleotide (e.g., a coding sequence) to control the initiation of transcription by RNA polymerase and expression of the coding sequence, such as one for the msr gene, msd gene, and/or the ret gene. Other transcriptional control regulatory elements (e.g., enhancer sequences, transcription factor binding sites) may also be operably linked to a gene if their location relative to a gene controls or regulates the expression of the gene.

Programmable Nuclease

As used herein, the term “programmable nuclease” is meant to refer to a polypeptide that has the property of selective localization to a specific desired nucleotide sequence target in a nucleic acid molecule (e.g., to a specific gene target) due to one or more targeting functions. Such targeting functions can include one or more DNA-binding domains, such as zinc finger domains characteristic of many different types of DNA binding proteins or TALE domains characteristic of TALEN proteins. Such targeting function may also include the ability to associate and/or form a complex with a guide RNA, which then localizes to a specific site on the DNA which bears a sequence that is complementary to a portion of the guide RNA (i.e., the spacer of the guide RNA). In some embodiments, the programmable nuclease may be a single protein which comprises both a domain that binds directly (e.g., a ZF protein) or indirectly (e.g., an RNA-guided protein) to a target DNA site, as well as a nuclease domain. In other embodiments, the programmable nuclease may be a composite of two or more separate proteins or domains (from different proteins) which together provide the necessary functions of selective DNA binding and nuclease activity. For example, the programmable nuclease may comprise a (a) nuclease-inactive RNA-guided nuclease (which still is capable of binding a guide RNA, localizing to a target DNA, and binding to the target DNA, but not capable of cutting or nicking the strands) fused to a (b) nuclease protein or domain, such as a FokI nuclease.

Promoter

As used herein, the term“promoter” is art-recognized and refers to a nucleic acid molecule with a sequence recognized by the cellular transcription machinery and which is able to initiate transcription of a downstream gene. A promoter can be constitutively active, meaning that the promoter is always active in a given cellular context, or conditionally active, meaning that the promoter is only active in the presence of a specific condition. For example, a conditional promoter may only be active in the presence of a specific protein that connects a protein associated with a regulatory element in the promoter to the basic transcriptional machinery, or only in the absence of an inhibitory molecule. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, may be used to drive expression by the various vectors of the present disclosure.

Recombinant Nucleic Acid

A “recombinant nucleic acid” or “recombinant nucleotide” refers to a molecule that is constructed by joining nucleic acid molecules, which optionally may self-replicate in a live cell.

Retron

As used herein, the term “retron” refers to a specific type of naturally-occurring and distinct DNA sequence found in the genome of many bacteria which typically encodes three distinct components, namely, (a) a non-coding RNA (“ncRNA”) (comprising contiguous inverted sequences (msr and msd), (b) a reverse transcriptase (RT)-coding gene (ret), and (c) in many cases, a retron-associated gene of unknown function. Retrons are particularly defined by their unique ability to produce a satellite DNA known as msDNA (multicopy single-stranded DNA). The ncRNA (comprising the msr and msd elements) and the ret gene are transcribed as a single polycitronic RNA transcript which processed into the ncRNA transcript and a transcript encoding the ret gene. The ncRNA then becomes folded into a specific secondary structure. Once translated, the RT then binds the folded ncRNA and reverse transcribes the msd region to form a single strand of cDNA (the msDNA) that remains covalently attached to the RNA template via a 2′-5′ phosphodiester bond and base-pairing between the 3′ ends of the msDNA and the RNA template. See FIG. 1A which provides a schematic of the production of an msDNA from a naturally-occurring retron.

Retron Component

As used herein, the term “retron component” refers to a distinct element or feature of a retron, namely (a) a non-coding RNA (“ncRNA”) (comprising contiguous inverted sequences (msr and msd), (b) a reverse transcriptase (RT)-coding gene (ret), and (c) in many cases, a retron-associated gene of unknown function.

RNA-Guided Nuclease

As used herein, an “RNA-guided nuclease” is a type of “programmable nuclease,” and a specific type of “nucleic acid-guided nuclease.” As used herein, the term “RNA-guided nuclease” or “RNA-guided endonuclease” refers to a nuclease that associates covalently or non-covalently with a guide RNA thereby forming a complex between the guide RNA and the RNA-guided nuclease. The guide RNA comprises a spacer sequence which comprises a nucleotide sequence having complementarity with a strand of a target DNA sequence. Thus, the RNA-guided nuclease is indirectly guided or programmed to localize to a specific site in a DNA molecule through its association with the guide RNA, which directly binds or anneals to a strand of the target DNA through its complementarity region via Watson-Crick base-pairing.

Sequence Identity

As used herein, the term “sequence identity” refers to the overall relatedness between polymeric molecules, e.g., between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). For example, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; each of which is incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna. CMP matrix. Methods commonly employed to determine percent identity between sequences include, but are not limited to those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988); incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software to determine homology between two sequences include, but are not limited to, GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990).

Subject

As used herein, the term“subject” refers to an individual organism, for example, an individual mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human primate. In some embodiments, the subject is a rodent. In some embodiments, the subject is a sheep, a goat, a cattle, a cat, or a dog. In some embodiments, the subject is a vertebrate, an amphibian, a reptile, a fish, an insect, a fly, or a nematode. In some embodiments, the subject is a research animal. In some embodiments, the subject is genetically engineered, e.g., a genetically engineered non-human subject. The subject may be of either sex and at any stage of development. The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein.

Stem

As used herein, the term “stem” refers to two or more base pairs, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more base pairs, formed by inverted repeat sequences connected at a “tip,” where the more 5′ or “upstream” strand of the stem bends to allows the more 3′ or “downstream” strand to base-pair with the upstream strand. The number of base pairs in a stem is the “length” of the stem. The tip of the stem is typically at least 3 nucleotides, but can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. Larger tips with more than 5 nucleotides are also referred to as a “loop.” An otherwise continuous stem may be interrupted by one or more bulges as defined herein. The number of unpaired nucleotides in the bulge(s) are not included in the length of the stem. The position of a bulge closest to the tip can be described by the number of base pairs between the bulge and the tip (e.g., the bulge is 4 bps from the tip). The position of the other bulges (if any) further away from the tip can be described by the number of base pairs in the stem between the bulge in question and the tip, excluding any unpaired bases of other bulges in between.

Synthetic or Artificial Nucleic Acid

A “synthetic or artificial nucleic acid” refers nucleic acids that are non-naturally occurring sequences. Such sequences do not originate from, or are not known to be present in any living organism (e.g., based on sequence search in existing sequence databases). Recombinant nucleic acids and synthetic nucleic acids also include those molecules that result from the replication of either of the foregoing. Engineered nucleic acid constructs of the present disclosure, such as the engineered retron described herein, may be encoded by a single molecule (e.g., encoded by or present on the same plasmid or other suitable vector) or by multiple different molecules (e.g., multiple independently-replicating vectors).

Target Site

As used herein, a “target site” as used herein is a polynucleotide (e.g., DNA such as genomic DNA) that includes a site or specific locus (“target site” or “target sequence”) targeted by a recombinant retron genome modification system disclosed herein. In the context of retron genome modification systems disclosed herein that comprise an RNA-guided nuclease, a target sequence is the sequence to which the guide sequence of a guide nucleic acid (e.g., guide RNA) will hybridize. For example, the target site (or target sequence) 5′-GTCAATGGACC-3′ within a target nucleic acid is targeted by (or is bound by, or hybridizes with, or is complementary to) the sequence 5′-GGTCCATTGAC-3′. Suitable hybridization conditions include physiological conditions normally present in a cell. For a double stranded target nucleic acid, the strand of the target nucleic acid that is complementary to and hybridizes with the guide RNA is referred to as the “complementary strand” or “target strand”; while the strand of the target nucleic acid that is complementary to the “target strand” (and is therefore not complementary to the guide RNA) is referred to as the “non-target strand” or “non-complementary strand.”

Treatment

As used herein, the terms“treatment,” “treat,” and“treating,” refer to a clinical intervention aimed to reverse, alleviate, delay the onset of, or inhibit the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed and/or after a disease has been diagnosed. In other embodiments, treatment may be administered in the absence of symptoms, e.g., to prevent or delay onset of a symptom or inhibit onset or progression of a disease. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to prevent or delay their recurrence.

Upstream and Downstream

As used herein, the terms “upstream” and “downstream” are terms of relativity that define the linear position of at least two elements located in a nucleic acid molecule (whether single or double-stranded) that is orientated in a 5′-to-3′ direction. A first element is said to be upstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 5′ to the second element. Conversely, a first element is downstream of a second element in a nucleic acid molecule where the first element is positioned somewhere that is 3′ to the second element.

Variant

As used herein the term“variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature, e.g., a variant retron RT is retron RT comprising one or more changes in amino acid residues as compared to a wild type retron RT amino acid sequence. The term“variant” encompasses homologous proteins having at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 99% percent identity with a reference sequence and having the same or substantially the same functional activity or activities as the reference sequence. The term also encompasses mutants, truncations, or domains of a reference sequence, and which display the same or substantially the same functional activity or activities as the reference sequence.

Vector

As used herein, the term “vector” permits or facilitates the transfer of a polynucleotide from one environment to another. It is a replicon such as a plasmid, phage, or cosmid into which another DNA segment may be inserted so as to bring about the replication of the inserted segment (e.g., the subject engineered retron). Generally, a vector is capable of replication when associated with the proper control elements. The term “vector” may include cloning and expression vectors, as well as viral vectors and integrating vectors.

Wild Type

As used herein the term “wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene, protein, or characteristic as it occurs in nature as distinguished from mutant or variant forms.

DETAILED DESCRIPTION

The present disclosure provides systems, methods and compositions used for precise genome editing, including installing nucleic acid insertions, replacements, and deletions at targeted and precise genome sites, wherein said systems, methods, and compositions are based on novel and/or modified retrons or components thereof, such as modified versions of the retron RTs of Table X, modified versions of the ncRNAs of Table A, and modified versions of the RTs of Table B.

In one aspect, the present disclosure provides recombinant retrons comprising one or more genetic modifications which improves the functionality and/or properties of a retron. Such genetic modifications can include a mutation, insertion, deletion, inversion, replacement, substitution, or translocation of one or more contiguous or non-contiguous nucleobases in a nucleic acid molecule encoding a retron or a component of a retron, such as an ncRNA or a reverse transcriptase. In various aspects, the retron that becomes modified with the one or more genetic modifications (i.e., the “pre-modified” or “unmodified” retron or retron component) is a naturally occurring retron or retron component (e.g., naturally occurring ncRNA of Table A or RT) ability to facilitate homology-dependent recombination (or HDR) in a cell, thereby resulting in a relative increase in the concentrations or amounts of msDNA comprising a DNA donor template. In particular embodiments, the recombinant retrons are based on and/or derived from a naturally-occurring retron, such as any retron-related sequence provided by Table X (the introduction of the one or more genetic modifications into a set of 7257 previously unknown retrons discovered through computational methods described herein (e.g., see Examples). In other embodiments, the recombinant retrons are based on introducing the one or more genetic modifications into previously available retron sequences (e.g., the “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 Dec. 2020, Pages 12632-12647” (incorporated herein by reference) to achieve recombinant retrons with the enhanced ability to produce increased concentrations or amounts of msDNA comprising a DNA donor template.

In another aspect, the present disclosure further provides nucleic acid molecules encoding the recombinant retrons and/or recombinant retron components (e.g., a recombinant ncRNA and/or a recombinant retron RT). In still another aspect, the present disclosure provides genome editing systems comprising recombinant retron components (e.g., recombinant ncRNA and/or recombinant RT), programmable nucleases (e.g., RNA-guided nucleases, such as CRISPR-Cas proteins, ZFPs, and TALENS), and guide RNAs (in the case where RNA-guide nucleases are used in said genome editing systems). In a further aspect, the disclosure provides nucleic acid molecules encoding the described genome editing systems and said components thereof, as well as polypeptides making up the components of said genome editing systems. In yet another aspect, the disclosure provides vectors for transferring and/or expressing said genome editing systems, e.g., under in vitro, ex vivo, and in vivo conditions. In still another aspect, the disclosure provides cell-delivery compositions and methods, including compositions for passive and/or active transport to cells (e.g., plasmids), delivery by virus-based recombinant vectors (e.g., AAV and/or lentivirus vectors), delivery by non-virus-based systems (e.g., liposomes and LNPs), and delivery by virus-like particles. Depending on the delivery system employed, the retron-based genome editing systems described herein may be delivered in the form of DNA (e.g., plasmids or DNA-based virus vectors), RNA (e.g., ncRNA and mRNA delivered by LNPs), a mixture of DNA and RNA, protein (e.g., virus-like particles), and ribonucleoprotein (RNP) complexes. Any suitable combinations of approaches for delivering the components of the herein disclosed retron-based genome editing systems may be employed. In one embodiment, each of the components of the retron-based genome editing system is delivered by an all-RNA system, e.g., the delivery of one or more RNA molecules (e.g., mRNA and/or ncRNA) by one or more LNPs, wherein the one or more RNA molecules form the ncRNA and guide RNA (as needed) and/or are translated into the polypeptide components (e.g., the RT and a programmable nuclease). In yet another aspect, the disclosure provides methods for genome editing by introducing a retron-based genome editing system described herein into a cell (e.g., under in vitro, in vivo, or ex vivo conditions) comprising a target edit site, thereby resulting in an edit at the target edit. In other aspects, the disclosure provides formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, recombinant cells and/or tissues modified by the recombinant retron-based genome modification systems and methods described herein, and methods of modifying cells by conducting genome editing and related DNA donor-dependent methods, such as recombineering, or cell recording, using the herein disclosed retron-based genome modification systems. The disclosure also provides methods of making the recombinant retrons, retron-based genome modification systems, vectors, compositions and formulations described herein, as well as to pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions that comprise the herein disclosed genome editing and/or modification systems.

Described herein are engineered retrons comprising one or more heterologous nucleic acids. The one or more heterologous nucleic acids may be inserted, for example, at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus. In some embodiments, the engineered retrons have structural improvements over their naturally existing counterparts or wild-type retrons at least with respect to the encoded ncRNA and/or the reverse transcriptase (RT), such that the engineered retron or the encoded ncRNA thereof, when delivered to a host cell, such as a mammalian host cell, exhibits various functional improvements over its naturally existing/wild-type retron elements.

Exemplary (non-limiting functional improvements) may include any one or more of the features described herein. For example, in some embodiments, the engineered retron may comprise a sequence modification (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: i) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulates (e.g., reduces) immunogenicity of the ncRNA encoded by the engineered retron (e.g., encoded by the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); iii) comprises a nucleotide sequence that modulates (e.g., inhibits or antagonizes) a function of the msDNA; and/or iv) modulates (e.g., improves) efficiency of targeted genomic engineering.

Thus, in general, the engineered retron is an engineered nucleic acid construct comprising: a) a first polynucleotide encoding a non-coding RNA (ncRNA), said first polynucleotide comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; and

b) one or more heterologous nucleic acids inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus.

The engineered nucleic acid construct (e.g., the engineered retron) may further comprise a second polynucleotide encoding a reverse transcriptase (RT), or a portion thereof, wherein the encoded RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.

In certain embodiments, the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in any one of Table C. In some embodiments, the RT does not comprise a polypeptide listed in Table X.

In certain embodiments, the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99⁰/a %, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A; and/or ii) a consensus polynucleotide sequence listed in Table C. In some embodiments, the polynucleotide encoding the RT does not comprise a polynucleotide of Table X.

In certain embodiments, the engineered retron of the invention encodes an ncRNA comprising: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA in Table B.

In certain embodiments, the engineered retron of the invention encodes an ncRNA and a reverse transcriptase (RT) or a functional domain thereof, wherein the ncRNA and the RT or functional domain thereof are as described above.

Specifically, in such embodiment, the ncRNA may comprise: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B.

Also in such embodiment, the reverse transcriptase (RT) or functional domain thereof comprises: (A) i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C; optionally, the RT does not comprise a polypeptide listed in Table X; OR (B) i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide in Table A; and/or optionally, the polynucleotide encoding the RT does not comprise a polynucleotide in Table X.

In certain embodiments, the engineered nucleic acid construct comprises: 1) an msr locus (that encodes the msr RNA portion of an msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retron reverse transcriptase (RT), wherein said msd RNA is capable of being reverse transcribed to form the msDNA by the retron reverse transcriptase (RT); and, 4) a heterologous nucleic acid inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus; wherein the engineered nucleic acid construct is engineered based on and/or to resemble a secondary structure of a wild-type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) any one of the sequences and/or structures as depicted in any one of SEQ ID NOs: of Table B and/or FIGS. 2-27 ; or b) a variant of a), having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: i) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; ii) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; iii) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides; wherein the ncRNA does not comprise an ncRNA associated with the sequences of Table X.

The engineered nucleic acid construct (e.g., the engineered retron) may comprise one or more sequence modifications (e.g., an insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: a) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); b) modulates (e.g., reduces) immunogenicity of ncRNA encoded by the engineered retron (e.g., the msr locus and/or the msd locus) in a host (e.g., a host comprising the mammalian cell); c) modulates (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or d) modulates (e.g., improves) efficiency of targeted genome editing/engineering.

In some embodiments, the engineered nucleic acid construct (e.g., the engineered retron) is engineered based on and/or to resemble a secondary structure of a wild-type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) the sequence of any one of Table B ncRNA sequences and/or the structure depicted in any one of FIGS. 2-27 ; or b) a variant of a), having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: i) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; ii) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; iii) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides.

Another aspect of the disclosure provides a vector system comprising a vector comprising the engineered retron described herein.

Another aspect of the disclosure provides an isolated host cell comprising the engineered retron described herein, or the vector system described herein.

Another aspect of the disclosure provides a pharmaceutical composition comprising the engineered retron described herein, or the vector system described herein.

Another aspect of the disclosure provides a delivery vehicle comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, the vector or vector system described herein, the host cell described herein, or the pharmaceutical composition described herein.

Another aspect of the disclosure provides a kit comprising the engineered retron described herein or the ncRNA encoded by the engineered retron described herein, and optionally instructions for genetically modifying a cell using the engineered retron described herein or the ncRNA encoded by the engineered retron described herein.

Another aspect of the disclosure provides a method of modifying a target DNA sequence in a host cell (e.g., a mammalian cell), the method comprising introducing into the host cell (e.g., the mammalian cell) the engineered retron of the invention, the ncRNA encoded by the engineered retron of the invention, or the vector/vector system described herein, to allow the production of the msDNA in the host cell (e.g., mammalian cell), wherein at least a part of the heterologous nucleic acid in the msDNA is integrated into the genome of the host (e.g., mammalian) cell at the target DNA sequence. Optionally, the target sequence is recognized by a suitable nuclease, such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE), and a double-stranded break (DSB) is created by the nuclease to facilitate/promote the insertion of the part of the heterologous nucleic acid into the target sequence. Further optionally, the target sequence modified/inserted by the part of the heterologous nucleic acid can no longer be recognized by the nuclease to re-create a DSB.

Another aspect of the disclosure provides a use of the engineered retron in the various methods described herein.

Another aspect of the disclosure provides a genome editing system comprising: a) nuclease capable of acting at a target site on a genome (e.g. human genome), such as a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE); and b) an engineered retron described herein, or an ncRNA encoded thereby, or a vector or a vector system comprising or encoding the same. Optionally, the nuclease may be linked to one or more element(s) of the engineered retron or the encoded ncRNA. For example, in one embodiment, the nuclease may be linked (e.g., fused or conjugated) to the reverse transcriptase of the engineered retron described herein. In another embodiment, the nuclease may engage/bind to form a complex with a nucleic acid guide sequence (such as a single-guided RNA of a Cas enzyme), wherein the guide sequence is linked to the ncRNA and/or msDNA of the engineered retron described herein.

Another aspect of the disclosure provides an enhanced genome editing system, comprising the genome editing system of the disclosure connected to a biomolecule that modulates host DNA repair, in order to, for example, modulate (e.g., enhance) the incorporation of the heterologous nucleic acid sequence into a genome (e.g., human genome).

With the general aspect of the disclosure described herein, specific aspects and embodiments of the disclosure are further described in the sections below. It should be understood that any one embodiment of the disclosure, including those described only in the examples or the claims, or only in one section herein below, can be combined with any one or more additional embodiments of the invention, unless such combination is expressly disclaimed or are improper.

A. Recombinant Retrons

The present disclosure provides engineered retrons, as well as compositions, systems, and methods that include or utilize the engineered retrons for genome modification, such as genome editing, cell recording, and recombineering.

Retrons were originally discovered in 1984 in Myxococcus xanthus bacterium when a short, multi-copy single-stranded DNA (msDNA) that is abundantly present in the bacterial cell was identified. Since then, a number of naturally existing retrons have been found in many prokaryotes such as bacteria.

As depicted in FIG. 1A, retrons encode and transcribe as a single RNA, which comprises a non-coding RNA (ncRNA) portion and a portion encoding a specialized reverse transcriptase (RT). The retron ncRNA (msr and msd) is the precursor of the hybrid molecule that eventually forms, and it initially folds into a typical RNA secondary structure that is recognized by the accompanying RT. The translated RT typically recognizes certain secondary structures in the ncRNA, and binds the RNA template downstream from the msd region. The RT initiates reverse transcription of the RNA towards its 5′ end, starting from the 2′-end of a conserved guanosine (G) residue found immediately after a double-stranded RNA structure (the a1/a2 region) within the ncRNA. A portion of the ncRNA serves as a template for reverse transcription, and reverse transcription terminates before reaching the msr locus. During reverse transcription, cellular RNase H degrades the segment of the ncRNA that serves as template, but not other parts of the ncRNA. The result of the reverse transcription, the msDNA, remains covalently attached to the RNA template via the 2′-5′ phosphodiester bond, and base-pairs with the RNA template using the 3′ end of the msDNA. See FIG. 1A for a general or typical organization of the retron coding sequence, including the RT encoding sequence and the msr and msd loci, as well as the synthesis of the msDNA by reverse transcription of the initial ncRNA transcript.

Many retrons also contain an accessory protein (not depicted in FIG. 1A), which may have a variable function that may not be fully understood. In certain embodiments, the engineered retrons described herein do not comprise the accessory protein naturally associated with the wild-type or template retron.

Applicant has discovered, analyzed, and phylogenetically classified 7257 previously unknown retrons from nature based on multiple criteria, including sequence homology and conserved predicted secondary structures, and has grouped these retrons into different phylogenetic clades based on sequence homology and/or conserved predicted secondary structures. These clades include Type IA_IIA1 (FIG. 2 ), Type 1B1 (FIG. 3 ), Type 1B2 (FIG. 4 ), Type 1C (FIG. 5 ), Type IIA1 other (FIG. 6 ), Type IIA2 (FIG. 7 ), Type IIA3 (FIG. 8 ), Type IIA4 (FIG. 9 ), Type IIA5 (FIG. 10 ), Type IIIA1 (FIG. 11 ), Type IIIA2 (FIG. 12 ), Type IIIA3 (FIG. 13 ), Type IIIA4 (FIG. 14 ), Type IIIA5 (FIG. 15 ), Type IIIunk (FIG. 16 ), Type IV (FIG. 1X), Type V (FIG. 19 ), Type VI (FIG. 20 ), Type XI Group 1 (FIG. 21 ), Type XI (Group 2) (FIG. 22 ), Type XII (FIG. 23 ), Type XIII (FIG. 24 ), Type XIV (FIG. 25 ), Eco107-like (FIG. 26 ), and Outgroup A (FIG. 27 ). The disclosure further describes the engineering and/or modification of these newly discovered retron sequences as a starting point to obtain useful recombinant retrons, such as those depicted in FIG. 1B.

FIG. 1B.1 depicts an embodiment of a recombinant retron construct (e.g., a nucleotide sequence cloned into an expression vector) contemplated by the present disclosure. In the top left schematic, the single thin black line represents a double-stranded nucleotide sequence (e.g., as cloned into an expression vector, such as a plasmid). The recombinant retron is constructed by modifying a starting point retron DNA sequence encoding a ncRNA (the msr/msd region) (such as any one of the herein disclosed 7257 newly discovered retron sequences, and specifically any one of the 7257 ncRNA sequences of Table B. A starting point retron DNA sequence encoding an ncRNA may be modified in any number of ways and can including one modification or more than one modification. For example, the retron DNA may modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100, or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or up to 2000 nucleotides substituted, inserted, or deleted from a starting point retron (e.g., a wildtype retron). Where more than one nucleotide of a starting point retron (e.g., a wildtype retron) is substituted, deleted, or inserted, the nucleotides may be contiguous or non-contiguous. While an engineered retron as a whole is not naturally-occurring, it may include components such as nucleotide sequences that do occur in nature. For example, an engineered retron can have nucleotide sequences from different organisms (e.g., from different bacteria species), or from completely synthetic/artificial/recombinant nucleic acid sequences. Thus, an engineered retron can have a bacterial nucleotide sequence, a human nucleotide sequence, a viral nucleotide sequence, and/or a synthetic/artificial/recombinant nucleotide sequence, and/or combinations of such sequences. An example of modifications of the recombinant retrons disclosed herein include the insertion of a heterologous nucleic acid sequence in a retron, for example, inserted into the ncRNA locus, such as in the msr or the msd loci. Linking guide RNA molecules to the 5′ and/or 3′ ends (i.e., linking one at the 5′ end of a ncRNA and/or one at the 3′ end of a ncRNA) also represents another modification contemplated by the recombinant retrons disclosed herein. In such embodiments, the guide RNA molecules may also be categorized or referred to more generally as types of heterologous nucleic acid sequences used to modify starting point retrons. These modifications are depicted in FIG. 1B.

In addition to the DNA encoding the ncRNA, the DNA encoding the RT may also be modified to obtain a recombinant RT. For example, the RT-encoding DNA may modified to contain at least one nucleotide modification, including a single nucleotide substitution, insertion, or deletion, or a substitution, insertion, or deletion of more than one nucleotide, i.e., up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or up to 100, or up to 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or up to 2000 nucleotides substituted, inserted, or deleted from a starting point retron (e.g., a wildtype retron) within the RT gene.

Such modifications to the DNA encoding ncRNA and/or RT may modulate the function of the ncRNA and/or RT in various ways, including i) modulating (e.g., enhancing) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulating (e.g., reducing) immunogenicity of ncRNA (msr locus and msd locus) encoded by the engineered retron in a host (e.g., a host comprising the mammalian cell); iii) modulating (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or iv) modulating (e.g., improving) efficiency of targeted genome editing/engineering.

In one embodiment, the present disclosure provides recombinant retrons having the general structure of: a) an msr locus; b) an msd locus encoding the msd RNA portion of the msDNA; c) a sequence encoding a retron reverse transcriptase (RT) (optionally in trans to the ncRNA), wherein the msd RNA is capable of being reverse transcribed (e.g., in a host cell such as a mammalian cell) to form an msDNA by the retron reverse transcriptase (RT); d) a heterologous nucleic acid (e.g., heterologous DNA) capable of being transcribed with the msr locus and/or the msd locus, optionally, the heterologous nucleic acid is inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus.

The engineered retrons of the invention are optionally structurally further modified to include one or more heterologous nucleic acids. The engineered retron may be further modified to provide various functional improvements, such as (without limitation), to enhance the production of msDNA in a cell (e.g., a mammalian cell, including a human cell).

In certain embodiments, the disclosure provides engineered retrons based on their conserved predicted secondary structures, such as those in FIGS. 2-27 .

In other embodiments, the disclosure provides engineered retrons based on their sequence identity. Exemplary RT amino acid sequences and ret gene nucleic acid sequences are provided in Table A. Exemplary RT consensus amino acid sequences and/or ret gene nucleic acid sequences are provided in Table C. Exemplary ncRNA sequences are provided in Table B.

Retron sequences provisioned out of the scope of the invention are provided in Table X.

In certain embodiments, exemplary engineered retrons of the invention (1) are engineered based on or engineered to resemble the secondary structures as depicted in any one of FIGS. 2-27 , and/or (2) are provided in Table B. Sequences with significant sequence percentage identity (e.g., at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity) are also within the scope of the invention.

In certain embodiments, the engineered nucleic acid construct comprises: 1) an msr locus (that encodes the msr RNA portion of an msDNA); 2) an msd locus encoding the msd RNA portion of the msDNA; 3) a sequence encoding a retron reverse transcriptase (RT), wherein said msd RNA is capable of being reverse transcribed to form the msDNA by the retron reverse transcriptase (RT); and, 4) a heterologous nucleic acid inserted at or within the msd locus, upstream of the msr locus, upstream or downstream of the msd locus; wherein the engineered nucleic acid construct is engineered based on and/or to resemble a secondary structure of a wild-type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: a) any one of the sequences and/or structures as depicted in any one of SEQ ID Nos of Table B and/or FIGS. 2-27 ; or b) a variant of a), having: i) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; ii) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or iii) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: i) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; ii) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; iii) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or iv) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides; wherein the ncRNA does not comprise an ncRNA associated with the sequences of Table X.

In certain embodiments, the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A. In some embodiments, the RT does not comprise a polypeptide identified in Table X.

In certain embodiments, the engineered retron of the invention encodes a reverse transcriptase (RT) or a functional domain thereof, comprising: i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide of Table A and/or ii) a consensus polynucleotide sequence listed in Table A. In some embodiments, the polynucleotide encoding the RT does not comprise a polynucleotide identified in Table X.

In certain embodiments, the engineered retron of the invention encodes an ncRNA comprising: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA of Table B.

In certain embodiments, the engineered retron of the invention encodes an ncRNA and a reverse transcriptase (RT) or a functional domain thereof, wherein the ncRNA and the RT or functional domain thereof are as described above.

Specifically, in such embodiments, the ncRNA may comprise: (I) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B; and wherein the ncRNA optionally excludes the ncRNA associated with the sequences identified in Table X.

Also in such embodiment, the reverse transcriptase (RT) or functional domain thereof comprises: (A) i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C; optionally, the RT does not comprise a polypeptide identified in Table X; OR (B) i) a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A; optionally, the polynucleotide encoding the RT does not comprise a polynucleotide associated with the sequences identified in Table X.

In certain embodiments, the heterologous nucleic acid is between >20 nucleotides and about 10,000 nucleotides.

The engineered retron may further comprise a sequence modification (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) in the msr locus and/or the msd locus that: i) modulates (e.g., enhances) reverse transcription, processivity, accuracy/fidelity, and/or production of the msDNA (e.g., in the mammalian cell); ii) modulates (e.g., reduces) immunogenicity of ncRNA (msr locus and msd locus) encoded by the engineered retron in a host (e.g., a host comprising the mammalian cell); iii) comprises a nucleotide sequence that modulates (e.g., inhibits, either permanently or transiently) a function of the msDNA; and/or iv) modulates (e.g., improves) efficiency of targeted genome editing/engineering.

Retron msr gene, msd gene, and RT nucleic acid sequences (e.g., the ret gene) as well as the encoded retron reverse transcriptase protein sequences that may serve as the template of the engineered retron described herein may be derived from any source, such as those in Table A, optionally excluding those associated with the sequences of Table X.

In some embodiments, template or wild-type (wt) sequences of the msr gene, msd gene, and the RT coding sequence (viz., the ret gene) used in the engineered retron are derived from a bacterial retron.

In some embodiments, representative template/wild-type retrons are from gram negative bacteria. In some embodiments, the retron is from a bacterium listed in Table X.

In some embodiments, the engineered retrons are engineered based on clades defined on retron/retron RTs, in which the retrons are associated with a tripartite system composed of the ncRNA, the RT and an additional protein or RT-fused domain with diverse enzymatic functions. See, for example, “Mestre et al., Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems, Nucleic Acids Research, Volume 48, Issue 22, 16 Dec. 2020, Pages 12632-12647” (incorporated herein by reference). While the clades are based primarily upon naturally occurring ncRNA and retron/retron RT, and an additional protein or RT-fused domain, the clades, for the purpose of serving as the templates for the subject engineered retrons, are not limited to naturally occurring sequences. Rather, the clades can also encompass non-naturally occurring ncRNA and RT, including, without limitation, recombinant, modified or altered, chimeric, hybrid, synthetic, artificial, etc.

Thus, according to the instant disclosure, retrons may be considered phylogenetically related based on a Neighbor-Joining algorithm of at least 75% (of at least 1000 replicates) and a Poisson correction distance measurement of no more than 0.05, based on alignment of the retron RT. Alternatively or in addition, retrons may be considered phylogenetically related when/if the same RT, or closely related RT, can recognize the secondary structures of the ncRNA of the retrons and reserve transcribe the retrons to produce msDNA.

In certain embodiments, sequence alignments between different retron sequences (e.g., ncRNA and/or RT (protein and/or nucleic acid) sequences) or secondary structure generations are based on software known to one of ordinary skill in the art.

The retron ncRNA sequences including msr and msd sequences within the same clade may be highly conserved at certain positions, while being less conserved at other positions.

Exemplary consensus sequences based on clade members were generated (see, for example, the corresponding FIGS. 2-27 , respectively) to show these conserved sequences and/or secondary structures, including the highly conserved nucleotides with at least 97% sequence conservation at red lettered-nucleotides, those with between 90-97% sequence conservation at black lettered-nucleotides, and 75-90% nucleotide sequence identity at grey-lettered nucleotides. Further structural limitations of the consensus sequences for the clades are provided as colored circles indicating the probability of having a base at that specific position, including red circles representing a base in 97% of the cases, black circles representing a base in 90-97% of the cases, and grey circles representing a base in 75-90% of the cases.

In some embodiments, the template ncRNA based on which the subject engineered retron is modified (including the msr and msd region sequences) is a consensus sequence for the various retron ncRNA (including msr and msd nucleic acid sequences) clades, as provided in any one of SEQ ID NOs: of Table B and the corresponding FIGS. 2-27 , respectively, including the bases that are highly conserved and depicted by a specific-colored letters or circles, and optionally further including bases that may be present at specific locations by specific-colored circles.

In some embodiments, the engineered retron is engineered, based on and/or to resemble a secondary structure of a wild-type or consensus retron encoding a wild-type or consensus retron ncRNA encompassed by: 1) the sequences and/or structures as depicted in any one of SEQ ID NOs: of Table B and the corresponding FIGS. 2-27 , respectively; or 2) a variant of 1), having: A) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; B) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or C) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides. Optionally, the variant of 1) further comprises: a) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; b) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; c) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or d) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides.

The engineered retron may be engineered by introducing the sequence modifications (e.g., deletions, additions, or substitutions) into the wild-type retron encoding wild-type retron ncRNA, or into the retron encoding the consensus retron ncRNA.

For example, a variant retron may not satisfy the sequence and/or structural requirements of any one of SEQ ID NOs: of Table B and the corresponding FIGS. 2-27 , respectively, but may still be a suitable template for the engineered retron described herein, so long as one or more of the conditions set forth in A)-C) and/or a)-d) are met.

In certain embodiments, the highly conserved sequences in the template retrons are preserved/conserved or substantially preserved/conserved in the engineered retron described herein.

In certain embodiments, all or substantially all the red lettered-nucleotides (i.e., those conserved in about 97% or more of the retrons in the same clade) are preserved/conserved in the engineered retron described herein. In certain embodiments, no more than 1, 2, or 3 (e.g., up to 1) nucleotide change(s) (e.g., deleted or substituted) occur per 10 red lettered-nucleotides in the engineered retron described herein. In certain embodiments, no more than about 0.3%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the red lettered-nucleotides are changed (e.g., deleted or substituted) in the engineered retron described herein.

In certain embodiments, all or substantially all the black lettered-nucleotides (i.e., those conserved in about 90-97% of the retrons in the same clade) are preserved/conserved in the engineered retron described herein. In certain embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., up to 1 or 2) nucleotide change(s) (e.g., deleted or substituted) occur per 10 black lettered-nucleotides are changed in the engineered retron described herein. In certain embodiments, no more than about 3%, 4%, 5% or 10% of the black lettered-nucleotides are changed (e.g., deleted or substituted) in the engineered retron described herein.

In certain embodiments, all or substantially all the grey lettered-nucleotides (i.e., those conserved in about 75-90% of the retrons in the same clade) are preserved/conserved in the engineered retron described herein. In certain embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (e.g., up to 3 or 4, or up to 7, 8, or 9) nucleotide change(s) (e.g., deleted or substituted) occur per 10 grey lettered-nucleotides are changed in the engineered retron described herein. In certain embodiments, no more than about 5%, 10%, 15%, 20%, or 25% of the grey lettered-nucleotides are changed (e.g., deleted or substituted) in the engineered retron described herein.

In certain embodiments, all or substantially all the red circled-nucleotides (i.e., those with a nucleotide in about 97% or more of the retrons in the same clade) are present in the engineered retron described herein. In certain embodiments, no more than 1, 2, or 3 (e.g., 0.3, 0.5, or up to 1) nucleotides are absent (e.g., deleted) per 10 red circled-nucleotides in the engineered retron described herein. In certain embodiments, 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides are present per 10 red circled-nucleotides in the engineered retron described herein. In certain embodiments, no more than about 0.3%, 0.5%, 1%, 2%, 3%, 4%, or 5% of the red circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein.

In certain embodiments, all or substantially all the black circled-nucleotides (i.e., those with a nucleotide in about 90-97% of the retrons in the same clade) are present in the engineered retron described herein. In certain embodiments, no more than 1, 2, 3, or 4 (e.g., up to 1 or 2) nucleotides are absent (e.g., deleted) per 10 black circled-nucleotides in the engineered retron described herein. In certain embodiments, 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides are present per 10 black circled-nucleotides in the engineered retron described herein. In certain embodiments, no more than about 1%, 2%, 3%, 5% or 10% of the black circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein.

In certain embodiments, all or substantially all the grey circled-nucleotides (i.e., those with a nucleotide in about 75-90% of the retrons in the same clade) are present in the engineered retron described herein. In certain embodiments, no more than 1, 2, 3, 4, or 5 (e.g., up to 2, 3, or 4) nucleotides are absent (e.g., deleted) per 10 grey circled-nucleotides in the engineered retron described herein. In certain embodiments, 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides are present per 10 grey circled-nucleotides in the engineered retron described herein. In certain embodiments, no more than about 5%, 10%, 15%, 20%, or 25% of the grey circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein.

In certain embodiments, all or substantially all the white circled-nucleotides (i.e., those with a nucleotide in about 50-75% of the retrons in the same clade) are present in the engineered retron described herein. In certain embodiments, no more than 1, 2, 3, 4, 5, 6, or 6 (e.g., up to 2, 3, 4, 5, 6) nucleotide are absent (e.g., deleted) per 10 white circled-nucleotides in the engineered retron described herein. In certain embodiments, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9 or 10) nucleotides are present per 10 grey circled-nucleotides in the engineered retron described herein. In certain embodiments, no more than about 5%, 10%, 15%, 20%, 30%, 40%, or 50% of the white circled-nucleotides are absent (e.g., deleted) in the engineered retron described herein.

In some embodiments, the engineered retron is synthetically produced. In other embodiments, the synthetically produced engineered retron comprises the sequences and/or secondary structures as depicted in any one of SEQ ID NOs: of Table B and the corresponding FIGS. 2-27 , respectively, and at least the conserved color lettered nucleotides according to their respective levels of sequence identity (e.g., red, black and gray letters), and/or at least the conserved colored circle nucleotides according to their respective levels of probability of sequence presence (e.g., red, black and gray circles).

In some embodiments, the sequence modification in the engineered retrons leads to/results in the encoded retron ncRNA having the desired functional improvement.

In certain embodiments, the one or more sequence modifications comprises, in the ncRNA, one or more of: (i) a modified (e.g., mutated, reduced, or eliminated) bulge in a1, a2, or both a1 and a2; (ii) an extension or shortening of a1, a2, or both a1 and a2; (iii) an extension or shortening of a spacer sequence between hairpin loops (e.g., S1, S2, S3, and/or S4 in FIG. 2 , or any of the S regions in an one of FIGS. 2-27 ); (iv) an additional or modified (e.g., mutated or eliminated) bulge in hairpin loops (e.g., L2 and/or L3 in FIG. 2 , or any of the L regions in an one of FIGS. 2-27 (e.g., by removing unpaired bases in the bulge, or by replacing unpaired bases with an equivalent number of base pairs)); (v) a modified (e.g., extended or shortened) length of hairpin loops (e.g., L1, L2, L3, and/or L4 in FIG. 2 , or any of the L regions in an one of FIGS. 2-27 ); (vi) an alternative L1 and/or L2 (in FIG. 2 , or any of the L regions in an one of FIGS. 2-27 ) having complement, reverse, or reverse complement sequences; (vii) a modified (e.g., increased) number of unpaired bases at the tip of hairpin loops (e.g., L1, L2, L3, and/or L4 in FIG. 2 , or any of the L regions in an one of FIGS. 2-27 ); (viii) a modified (e.g., increased or decreased) GC content in hairpin loops (e.g., L1, L2, L3, and/or L4 in FIG. 2 , or any of the L regions in an one of FIGS. 2-27 ); (ix) an insertion of the heterologous nucleic acid in spacer sequences between hairpin loops (e.g., S1, S2, S3 and/or S4 in FIG. 2 , or any of the S regions in an one of FIGS. 2-27 ), or at the tip of hairpin loops (e.g., L1, L2, L3, and/or L4 in FIG. 2 , or any of the L regions in an one of FIGS. 2-27 ); (x) a deletion of one or more hairpin loops (e.g., L1, L2, L3 and/or L4 in FIG. 2 , or any of the L regions in an one of FIGS. 2-27 ); (xi) an addition of a new loop in a spacer sequence between hairpin loops (e.g., S1, S2, S3, and/or S4 in FIG. 2 , or any of the S regions in an one of FIGS. 2-27 ); (xii) circularization of the ncRNA with the 5′ end and the 3′ end of the ncRNA being connected either directly, or via a spacer sequence; (xiii) a repositioned branching guanosine capable of initiating reverse transcription priming; (xiv) a staggered end sequence that reduces immunogenicity of the retron ncRNA, created by, e.g., adding or removing the 5′ a1 nucleotides and/or the 3′ a2 nucleotides; and/or, (xv) an antisense sequence complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid, wherein the antisense sequence hybridizes to and inhibits said gRNA in the encoded retron ncRNA, and wherein said antisense sequence is removed upon reverse transcription of the msDNA.

Unless specifically indicated otherwise, the a1 and a2 regions are both single-stranded and substantially reverse complementary to each other, forming a stem with optional interruption by a symmetric or asymmetric bulge, with optional one or more 5′ and/or 3′ overhang/unpaired nucleotide(s), wherein the a1 region generally ends before (e.g., ends immediately 5′ to) the conserved branching guanosine (G) providing the 2′-OH for reverse transcription priming.

In some embodiments, the sequence change comprises a mutated, reduced, or eliminated bulge in the a1/a2 stem region, including sequence change(s) in one (i.e., a1 or a2) strand, or both a1 and a2 strands.

For example, in some embodiments, the sequence change comprises deleting nucleotides from a1, a2, or both a1 and a2, such that the size of the bulge is reduced, or a symmetrical bulge becomes asymmetrical or vice versa, or a bulge is eliminated.

In some embodiments, the sequence change comprises replacing/substituting nucleotides in a1, a2, or both a1 and a2, such that previously unpaired bases in the bulge become base-paired.

In some embodiments, the sequence change comprises replacing an unpaired purine base with one or more unpaired pyrimidine base(s).

In some embodiments, the sequence change comprises replacing an unpaired pyrimidine base with one or more unpaired purine base(s).

In some embodiments, the sequence change comprises replacing one unpaired purine base (e.g., A or G) with another unpaired purine base (e.g., G or A, respectively).

In some embodiments, the sequence change comprises replacing one unpaired pyrimidine base (e.g., T/U or C) with another unpaired pyrimidine base (e.g., C or T/U, respectively).

In some embodiments, the sequence change comprises an extension or shortening of a1, a2, or both a1 and a2.

For example, the length of a1 can be shortened by deleting 5′ overhang, deleting any upstream bulge nucleotides, deleting bases involved in base-pairing. Likewise, the length of a1 can be extended by adding 5′ overhang, adding any upstream bulge nucleotides, adding bases involved in base-pairing.

In some embodiments, the length of a2 can be shortened by deleting 5′ overhang, deleting any downstream bulge nucleotides, deleting bases involved in base-pairing. Likewise, the length of a2 can be extended by adding 5′ overhang, adding any downstream bulge nucleotides, adding bases involved in base-pairing.

In some embodiments, the spacer sequences between hairpin loops as depicted in any one of FIGS. 2-27 , (e.g., S1, S2, S3 and/or S4 in FIG. 2 ) can be extended or shortened. In some embodiments the modification can be by inserting a heterologous nucleic acid sequence in spacer sequences between hairpin loops (e.g., S1, S2, S3 and/or S4 in FIG. 2 ). In certain embodiments, one or more heterologous nucleic acid sequences is inserted in a spacer sequence in the msd region. In some embodiments, the modification of the spacer region can be by interrupting the spacer with additional bulges or hairpin loops.

In other embodiments, a bulge in the hairpin loops are mutated or eliminated (e.g., by removing unpaired bases) in the bulge, such that, for example, a symmetric bulge becomes an unsymmetrical bulge, or an unsymmetrical bulge becomes a symmetric one or an even more unsymmetrical one. In certain embodiments, unpaired bases in the bulge is replaced with an equivalent number of base pairs. The additional base pairs may be merged into the stem at one or both ends of the previous bulge, or may bisect a previous bulge to create two bulges.

In some embodiments, the length of one or more hairpin loops as depicted in any one of FIGS. 2-27 , (e.g., L1, L2, L3 and/or L4 of FIG. 2 ) can be extended or shortened. For example, the number of unpaired bases within the tip or loop can be increased or decreased. Further, a heterologous nucleic acid sequence of interest can be inserted within the tip or the hairpin loop. In certain embodiments, the heterologous nucleic acid sequence of interest is inserted within the tip or the hairpin loop in the msd locus.

In other embodiments, the GC content in the tip or hairpin loops are increased or decreased.

In still other embodiments, a hairpin loop can be deleted.

In some embodiments, the ncRNA with the 5′ end and the 3′ end of the ncRNA can be circularized by being connected either directly, or via a spacer sequence.

In some embodiments, one or more hairpin loops (e.g. L1, L2, L3 and/or L4 of FIG. 2 ) are modified to have complement, reverse, or reverse complement sequences.

In certain embodiments, the branching guanosine (G) capable of initiating reverse transcription priming is repositioned. For example, the G can be placed further downstream of the end of the a1 sequence by, for example, 1, 2, 3, 4, or 5 additional nucleotides.

In certain embodiments, immunogenicity of the retron ncRNA is reduced by, e.g., adding or removing the 5′ a1 nucleotides and/or the 3′ a2 nucleotides.

In certain embodiments, the one or more heterologous nucleic acid sequences (inserted into the subject engineered retron) comprise: a) a heterologous nucleic acid (such as the coding sequence for an RNA aptamer or a ribozyme) inserted into the msr locus or the msd locus (such as in an S region (e.g., S1, S2, S3 and/or S4 in FIG. 2 , or any of the S regions in any one of FIGS. 2-27 ), or the tip of an L region (e.g., L1, L2, L3 and/or L4 in FIG. 2 , or any of the L regions in any one of FIGS. 2-27 ), or upstream or downstream of either the msr locus or the msd locus; or b) a first heterologous nucleic acid inserted into the msd locus, and a second heterologous nucleic acid inserted either upstream of the msr locus or downstream of the msd locus, wherein the second heterologous nucleic acid encodes a CRISPR/Cas guide RNA (gRNA).

In certain embodiments, an antisense sequence complementary to a CRISPR/Cas guide RNA (gRNA) sequence encoded by the heterologous nucleic acid can be included, wherein the antisense sequence hybridizes to and inhibits the gRNA in the encoded retron ncRNA, and wherein the antisense sequence is removed upon reverse transcription of the msDNA.

In certain embodiments, said heterologous nucleic acid encodes a protein or peptide of interest, or wherein said heterologous nucleic acid comprises or encodes a donor/template sequence (e.g., a donor that corrects/repairs/removes a mutation at the target genome site, such as a mutated exon in a disease gene; a functional DNA element (such as a promoter, an enhancer, a protein binding sequence, a methylation site, a homology region for assisting gene editing, etc.); or a coding sequence for a functional RNA element (ncRNAs, etc.)).

In certain embodiments, the protein or peptide of interest comprises a therapeutic protein (such as a wildtype protein defective in a disease cell, or a therapeutic antibody or antigen-binding fragment thereof) useful in treating a disease.

Other heterologous nucleic acids of the invention are described in other section of the specification, all incorporated herein by reference.

In some embodiments, the template/wild-type retron for the engineered retron encodes a wild-type or consensus retron ncRNA polynucleotide having a consensus secondary structure shown in any one of FIGS. 2-27 , which are described individually below:

Variants of this template, which can also be used in the engineered retron of the invention, include a variant having: A) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; B) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or C) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: a) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; b) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; c) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or d) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides.

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in SEQ ID NO. XX and FIGS. 2-27 .

In some embodiments, the non-coding RNA (ncRNA) portion of the engineered retron comprises a polynucleotide (e.g., a DNA molecule) encoding an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B. In some embodiments, the ncRNA does not comprise an ncRNA associated with the sequences of Table X.

Amplification of an engineered retron described herein may be performed, for example, before transfection of cells or ligation into vectors. Any method for amplifying the engineered retron may be used, including, but not limited to polymerase chain reaction (PCR), isothermal amplification, nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR). In one embodiment, the engineered retron comprise common 5′ and 3′ priming sites to allow amplification of retron sequences in parallel with a set of universal primers. In another embodiment, a set of selective primers is used to selectively amplify a subset of retron sequences from a pooled mixture.

In some embodiments, the template/wild-type retron for the engineered retron encodes a wild-type or consensus retron ncRNA polynucleotide having a consensus secondary structure shown in FIG. 2-27 , and as described individually below:

Type IA/IA1 retron (FIG. 2 )

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-L2-S2-L3-S3-a2-S4-L4,

wherein: a1/a2 is a stem 8 bp in length; L1 is a stem of 5 bps with a 10-nt tip; L2 is a stem of 7 bps with a 5-nt tip, and a 1/1 bulge 3 nt from the tip; L3 is a stem of 23 bps with a 22-nt tip, and a 2/2 bulge 21 bps from the tip; L4 is a stem of 11 bps with a 5-nt tip; S1 is a single-stranded spacer region between the a1/a2 stem and L1, with no spacer between L1 and L2; S2 is a single-stranded spacer region between L2 and L3; S3 is a single-stranded spacer region between L3 and the a1/a2 stem; and S4 is a single-stranded spacer region between the a1/a2 stem and L4, and the conserved nucleotides are as shown in SEQ ID NO: 1 and FIG. 2 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 2 .

Type IB1 Retron (FIG. 3)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-a2,

wherein: a1/a2 is a stem 6 bps in length with a 2/2 bulge 3 bps from the tip, wherein a1 has a 2-nt overhang and a2 has a 6-nt overhang; L1 is a stem of 14 bps with a 3-nt tip, a 1/0 bulge 4 bps from the tip, and a 0/6 bulge 10 bps from the tip; L2 is a stem of 23 bps with a 5-nt tip, a 1/1 bulge 4 bps from the tip, and a 0/1 bulge 18 bps from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and a1/a2, the conserved nucleotides are as shown in FIG. 3 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 3 .

Type IB2 Retron (FIG. 4)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-L2-S2-L3-S3-L4-S4-a2,

wherein: the a1/a2 stem is 16 bp in length, with a 17-base 5′ overhang and a 16-base 3′ overhang; L1 is a stem of 6 bps with a 4-nt tip; L2 is a stem of 4 bps with a 4-nt tip, with a 2/2 bulge 2 nts from the tip; L3 is a stem of 3 bps with a 5-nt tip; L4 is a stem of 9 bps with a 5-nt tip, and a 1/1 bulge 4 nts from the tip; S1, S2, S3, and S4 are single-stranded spacer regions between the a1/a2 stem and L1, L2 and L3, L3 and L4, and L4 and the a1/a2 stem, respectively, with no spacer between L1 and L2; wherein the last 5 nts of S1 and the 5^(th)-9^(th) nts of S2 form a 5-bp stem, and, the conserved nucleotides are as shown in FIG. 4 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 4 .

Type 1C Retron (FIG. 5)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-a2,

wherein: a1/a2 is a stem 13 bps in length; L1 is a stem of 9 bps with a 3-nt tip; L2 is a stem of 10 bps with a 5-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and a1/a2, the conserved nucleotides are as shown in FIG. 5 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 5 .

Type IIA1 Retron (FIG. 6)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-a2,

wherein: a1/a2 is a stem 10 bps in length with a 1-nt overhang on a2; L1 is a stem of 10 bps with a 3-nt tip; L2 is a stem of 7 bps with a 5-nt tip; L3 is a stem of 27 bps with a 8-nt tip and a 0/2 bulge 26 bps from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3; S4 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in FIG. 6 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 6 .

Type IIA2 Retron (FIG. 7)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-L3-S3-a2,

wherein: a1/a2 is a stem 7 bp in length with no overhangs; L1 is a stem of 8 bps with a 3-nt tip; L2 is a stem of 30 bps with a 8-nt tip, a 1/1 bulge 2 bps from the tip, and a 1/1 bulge 27 bps from the tip; L3 is a stem of 8 bps with a 5-nt tip, and a 0/1 bulge 3 nt from the tip; S1 is a single-stranded spacer region between the a1/a2 stem and L1; S2 is a single-stranded spacer region between L1 and L2; and, S3 is a single-stranded spacer region between L3 and the a1/a2 stem; the conserved nucleotides are as shown in FIG. 7 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 7 .

Type IIA3 Retron (FIG. 8)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-L2-S2-L3-S3-a2,

wherein: a1/a2 is a stem 6 bps in length; L1 is a stem of 8 bps with a 9-nt tip; L2 is a stem of 8 bps with a 3-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L2 and L3; S3 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in FIG. 8 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 8 .

Type IIA4 Retron (FIG. 9)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-a2-L1-S1-L2-L3-S2-L4-S3,

wherein: a1/a2 is a stem 3 bp in length with no overhangs and a 7-nt tip; L1 is a stem of 7 bps with a 3-nt tip; L2 is a stem of 6 bps with a 4-nt tip; L3 is a stem of 40 bps with a 5-nt tip, and a 2/2 bulge 3 bps from the tip, a 5/4 bulge 10 bps from the tip, and a 12/15 bulge 30 bps from the tip; L4 is a stem of 4 bps with a 9-nt tip; S1 is a single-stranded spacer region between L1 and L2/L3; S2 is a single-stranded spacer region between L2/L3 and L4; S3 is a single-stranded spacer region between L4 and the 3′ end of the ncRNA; and the conserved nucleotides are as shown in FIG. 9 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 9 .

Type IIA5 Novel Retron (FIG. 10)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-a2,

wherein: a1/a2 is a stem 15 bps in length with a 1-nt overhang on a1, a 13-nt overhang on a2, and a 7/5 bulge 13-nt from the tip; L1 is a stem of 10 bps with a 3-nt tip; L2 is a stem of 35 bps with a 3-nt tip; L3 is a stem of 6 bps with a 5-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3; S4 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in FIG. 10 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 10 .

Type IA1 Retron (FIG. 11)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-L2-S2-L3-S3-a2,

wherein: a1/a2 is a stem 2 bps in length with a 1-nt overhang on a2; L1 is a stem of 8 bps with a 4-nt tip; L2 is a stem of 9 bps with a 3-nt tip and a 1/1 bulge 3 bps from the tip; L3 is a stem of 20 bps with a 3-nt tip and a 1/2 bulge 3 bps from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L2 and L3; S3 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in FIG. 11 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 11 .

Type IIIA2 Retron (FIG. 12)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-L4-S5-a2,

wherein: a1/a2 is a stem 15 bp in length; L1 is a stem of 6 bps with a 4-nt tip; L2 is a stem of 13 bps with a 5-nt tip; L3 is a stem of 4 bps with a 8-nt tip; L4 is a stem of 20 bps with a 4-nt tip and a 2/2 bulge 6 bp from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3; S4 is a single-stranded spacer region between L3 and L4; S5 is a single-stranded spacer region between L4 and a1/a2; the conserved nucleotides are as shown in FIG. 12 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 12 .

Type IA3 Retron (FIG. 13)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-L4-L5-L6-S5-a2,

wherein: a1/a2 is a stem 24 bps in length and having a 1/0 bulge 15 bps from the tip, and a 1/1 bulge 19 bps from the tip; L1 is a stem of 7 bps with a 4-nt tip; L2 is a stem of 9 bps with a 8-nt tip; L3 is a stem of 8 bps with a 4-nt tip; L4 is a stem of 4 bps with a 9-nt tip, and a 2/2 bulge 3 bps from the tip; L5 is a stem of 19 bps with a 18-nt tip; L6 is a stem of 5 bps with a 3-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3; S4 is a single-stranded spacer region between L3 and L4; S5 is a single-stranded spacer region between L6 and a1/a2; the conserved nucleotides are as shown in FIG. 13 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90/6 of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 13 .

Type IA4 Retron (FIG. 14)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-a2,

wherein: a1/a2 is a stem 5 bps in length with a 1/2 bulge 2 bps from the tip; L1 is a stem of 8 bps with a 6-nt tip; L2 is a stem of 8 bps with a 5-nt tip; L3 is a stem of 13 bps with a 14-nt tip and a 1/0 bulge 2 bps from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3: S4 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in SEQ ID NO: 19 and FIG. 20 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 14 .

Type IIIA5 Retron (FIG. 15)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-L3-L4-S3-a2,

wherein: the a1/a2 stem is 11 bp in length with no overhang; L1 is a stem of 9 bps with a 3-nt tip; L2 is a stem of 14 bps with a 5-nt tip; L3 is a stem of 9 bps with a 7-nt tip; L4 is a stem of 15 bps with a 7-nt tip; S1, S2, and S3 are single-stranded spacer regions between the a1/a2 stem and L1, L2 and L3, and L4 and the a1/a2 stem, respectively, with no spacer between L1 and L2, and no spacer between L2 and L3; wherein the 5^(th)-2^(nd) last nts of S2 and the 3^(rd)-6^(th) nts of S3 forms a 4-bp stem, and, the conserved nucleotides are as shown in FIG. 15 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 15 .

Type IIIunk Retron (FIG. 16)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-a2,

wherein: a1/a2 is a stem 11 bps in length; L1 is a stem of 12 bps with a 2-nt tip; L2 is a stem of 21 bps with a 1-nt tip; L3 is a stem of 20 bps with a 4-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3; S4 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in FIG. 16 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90/6 of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 16 .

Type IV Retron (FIG. 17)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-L2-S2-L3-S3-a2,

wherein: a1/a2 is a stem 9 bp in length with no overhang; L1 is a stem of 5 bps with a 6-nt tip; L2 is a stem of 9 bps with a 4-nt tip; L3 is a stem of 26 bps with a 5-nt tip, a 0/1 bulge 7 bps from the tip, and a 0/1 bulge 9 bps from the tip; S1 is a single-stranded spacer region between the a1/a2 stem and L1, with no spacer region between L1 and L2; S2 is a single-stranded spacer region between L2 and L3; and, S3 is a single-stranded spacer region between L3 and the a1/a2 stem; the conserved nucleotides are as shown in FIG. 17 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 17 .

Type IX Retron (FIG. 18)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-L1-S1-L2-S2-a2,

wherein: a1/a2 is a stem 12 bp in length, wherein a1 has a 14-nt overhang and a2 has a 2-nt overhang; L1 is a stem of 11 bps with a 3-nt tip and a 1/3 bulge 7 bp from the tip; L2 is a stem of 25 bps with a 7-nt tip; S1 is a single-stranded spacer region between L1 and L2; S2 is a single-stranded spacer region between L2 and a1/a2; the conserved nucleotides are as shown in FIG. 18 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 18 .

Type V Retron (FIG. 19)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-a2,

wherein: a1/a2 is a stem 13 bps in length; L1 is a stem of 20 bps with a 4-nt tip and a 6/4 bulge 6 bps from the tip; L2 is a stem of 14 bps with a 4-nt tip and a 1/0 bulge 5 bps from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and a1/a2, the conserved nucleotides are as shown in FIG. 19 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 19 .

Type VI Retron (FIG. 20)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-L4-S4-a2,

wherein: a1/a2 is a stem 4 bp in length with a 1 bp 5′ overhang; L1 is a stem of 7 bps with a 4-nt tip; L2 is a stem of 8 bps with a 4-nt tip; L3 is a stem of 16 bps with a 6-nt tip, a 3/4 bulge 3 bps from the tip, a 2/3 bulge 5 bps from the tip, and a 3/1 bulge 8 bps from the tip; S1 is a single-stranded spacer region between the a1/a2 stem and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3, with no spacer region between L3 and L4; S4 is a single-stranded spacer region between L4 and the a1/a2 stem; and, the conserved nucleotides are as shown in FIG. 20 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 20 .

Type XI Group 1 Retron (FIG. 21)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-a2,

wherein: a1/a2 is a stem 16 bps in length with a 5-nt overhang on a1, and a 3-nt overhang on a2; L1 is a stem of 9 bps with a 3-nt tip; L2 is a stem of 7 bps with a 13-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and a1/a2, the conserved nucleotides are as shown in FIG. 21 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 21 .

TYPE XI Group 2 retron (FIG. 22 )

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-a2,

wherein: a1/a2 is a stem 13 bps in length with a 1-nt overhang on a2; L1 is a stem of 7 bps with a 3-nt tip and a 2/2 bulge 1 bp from the tip; L2 is a stem of 8 bps with a 20-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and a1/a2, the conserved nucleotides are as shown in FIG. 22 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 22 .

Type XII Retron (FIG. 23)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-a2,

wherein: a1/a2 is a stem 13 bps in length with a 1-nt overhang on a2; L1 is a stem of 7 bps with a 3-nt tip, and a 2/2 bulge 1 bp from the tip; L2 is a stem of 8 bps with a 19-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and a1/a2, the conserved nucleotides are as shown in FIG. 23 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 23 .

Type XIII Retron (FIG. 24)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-L3-S3-a2,

wherein: a1/a2 is a stem 7 bp in length with no overhangs; L1 is a stem of 8 bps with a 3-nt tip; L2 is a stem of 30 bps with a 8-nt tip, a 1/1 bulge 2 bps from the tip, and a 1/1 bulge 27 bps from the tip; L3 is a stem of 8 bps with a 5-nt tip, and a 0/1 bulge 3 nt from the tip; S1 is a single-stranded spacer region between the a1/a2 stem and L1; S2 is a single-stranded spacer region between L1 and L2; and, S3 is a single-stranded spacer region between L3 and the a1/a2 stem; the conserved nucleotides are as shown in FIG. 24 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 24 .

Type XIV Retron (FIG. 25)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-L2-S2-L3-S3-a2,

wherein: the a1/a2 stem is 15 bp in length with no overhang, and a 4/2 bulge 7 bps from the 5′ end of a1; L1 is a stem of 8 bps with a 5-nt tip; L2 is a stem of 7 bps with a 5-nt tip; L3 is a stem of 13 bps with a 2-nt tip, a 5/9 bulge 5 bps from the tip, and a 5/5 bulge 8 bps from the tip; S1, S2, and S3 are single-stranded spacer regions between the a1/a2 stem and L1, L2 and L3, and L3 and the a1/a2 stem, respectively, with no spacer between L1 and L2; and wherein the 5^(th)-3^(rd) last nts of S1 and the 2nd-5th 4 nts of S2 forms a 3-bp stem; and, the conserved nucleotides are as shown in FIG. 25 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 25 .

Ec107-Like Retron (FIG. 26)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-a2,

wherein: a1/a2 is a stem 12 bp in length; L1 is a stem of 4 bps with a 8-nt tip; L2 is a stem of 8 bps with a 3-nt tip; L3 is a stem of 22 bps with a 3-nt tip, a 4/6 bulge 6 bp from the tip, a 3/3 bulge 13 bp from the tip, and a 1/1 bulge 18 bp from the tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3; and S4 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in FIG. 26 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 26 .

Outgroup A Retron (FIG. 27)

In some embodiments, the template/wt retron for the subject engineered retron encodes a wild-type retron ncRNA polynucleotide having a consensus secondary structure that can be described as:

a1-S1-L1-S2-L2-S3-L3-S4-a2,

wherein: a1/a2 is a stem 5 bps in length with a 2-nt overhang on a2; L1 is a stem of 11 bps with a 4-nt tip; L2 is a stem of 8 bps with a 3-nt tip; L3 is a stem of 18 bps with a 3-nt tip; S1 is a single-stranded spacer region between a1/a2 and L1; S2 is a single-stranded spacer region between L1 and L2; S3 is a single-stranded spacer region between L2 and L3; S4 is a single-stranded spacer region between L3 and a1/a2, the conserved nucleotides are as shown in FIG. 27 , and wherein the colored circled nucleotides are present at the respective levels of certainty (e.g., at least about 97% of the red-circled nucleotides, at least about 90-97% of the black-circled nucleotides, at least about 75-90% of the grey-circled nucleotides, and at least about 50% of the white-circled nucleotides are present).

In some embodiments, the engineered retron is entirely synthetically produced and having the conserved nucleotides as denoted by the colored letters as shown in FIG. 27 .

Amplification of an engineered retron described herein (e.g., in FIGS. 2-27 ) may be performed, for example, before transfection of cells or ligation into vectors. Any method for amplifying the engineered retron may be used, including, but not limited to polymerase chain reaction (PCR), isothermal amplification, nucleic acid sequence-based amplification (NASBA), transcription mediated amplification (TMA), strand displacement amplification (SDA), and ligase chain reaction (LCR). In one embodiment, the engineered retron comprise common 5′ and 3′ priming sites to allow amplification of retron sequences in parallel with a set of universal primers. In another embodiment, a set of selective primers is used to selectively amplify a subset of retron sequences from a pooled mixture.

Variants of these templates, which can also be used in the engineered retron of the invention, include a variant having: A) up to 1, 2, or 3 (e.g., up to 1) nucleotide changes per 10 red lettered-nucleotides; B) up to 4, 5, or 6 (e.g., up to 1 or 2) nucleotide changes per 10 black lettered-nucleotides; and/or C) up to 7, 8, or 9 (e.g., up to 3 or 4) nucleotide changes per 10 grey lettered-nucleotides; and/or optionally further comprising: a) 7, 8, 9, or 10 (e.g., 9 or 10) nucleotides present per 10 red-circled nucleotides; b) 6, 7, 8, 9, or 10 (e.g., 8, 9 or 10) nucleotides present per 10 black-circled nucleotides; c) 4, 5, 6, 7, 8, 9, or 10 (e.g., 6, 7, 8, 9 or 10) nucleotides present per 10 grey-circled nucleotides; and/or d) 2, 3, 4, 5, 6, 7, 8, 9, or 10 (e.g., 4, 5, 6, 7, 8, 9, or 10) nucleotides present per 10 white-circled nucleotides.

In some embodiments, the non-coding RNA (ncRNA) portion of the engineered retron comprises a polynucleotide (e.g., a DNA molecule) encoding an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B. In some embodiments, the ncRNA does not comprise an ncRNA associated with the RT sequences of Table X.

B. Heterologous Nucleotide Sequence (HNS)

The engineered retron may comprise or encodes a heterologous nucleic acid (e.g., DNA or RNA) within the msr locus or the msd locus (such as in an S region or the tip of an L region in the consensus structure in any one of FIGS. 2-27 , and variants thereof), or upstream or downstream of either the msr locus or the msd locus. In some embodiments, the heterologous nucleic acid is inserted within the msd locus. In some embodiments, the heterologous nucleic acid is inserted upstream of the msr locus. In some embodiments, the heterologous nucleic acid is inserted upstream or downstream of the msd locus. In other embodiments, the heterologous nucleic acid is inserted in spacer sequences (e.g., S1, S2, S3 and/or S4 as depicted in FIG. 2 ) between hairpin loops and/or within hairpin loops (e.g., L1, L2, L3 and/or L4 as depicted in FIG. 2 ), e.g., the tip or loop region, or within a bulge. In some embodiments, the heterologous nucleic acid sequence can be inserted in the tip of an L region or hairpin loop (e.g., L1, L2, L3 and/or L4 as depicted in FIG. 2 ). In some embodiments, one or more heterologous nucleic acids are inserted into the msd locus. In some embodiments, a first heterologous nucleic acid is inserted into the msd locus, and a second heterologous nucleic acid is inserted either upstream of the msr locus or upstream or downstream of the msd locus.

In some embodiments, the heterologous nucleic acid comprises flanking contiguous nucleotides (also referred to as homologous arms) that are substantially complementary to a target site genomic sequence of a cell to facilitate insertion of at least part of the heterologous nucleic acid into the genome of the cell at the target site via homology directed repair (HDR). In some embodiments, the heterologous nucleic acid is between >20 nucleotides and 10,000 nucleotides (e.g., including the flanking homologous arms).

In some embodiments, one or both of the homology arm(s) on the heterologous nucleic acid are 100% identical to a target genomic sequence. In some embodiments, one or both of the homology arm(s) on the heterologous nucleic acid are less than 100% complementary to the target genomic sequence, for example, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to a target genomic sequence.

The part of the heterologous nucleic acid to be inserted into the target genome sequence is sometimes referred to as a donor sequence. The donor sequence may be partially identical to the full length or portions thereof of a target genomic sequence, or may be unrelated to the target genomic sequence. The donor sequence may be used, for example, to introduce modifications (e.g., substitutions, deletions, insertions, or a combination thereof) such as mutations or other genetic changes (e.g., genetic elements such as stop codons or a shift in an open reading frame on the target polynucleotide) into its target sequence.

In some embodiments, the heterologous nucleic acid sequence is or encodes a biologically active molecule such as, but not limited to, a therapeutic protein.

Any therapeutic proteins may be encoded by the heterologous nucleic acid.

In some embodiments, the heterologous nucleic acid sequences encodes one or more prophylactically- or therapeutically-active proteins, polypeptides, or other factors.

As a non-limiting example, the heterologous sequences may be or encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer. As another non-limiting example, the heterologous sequences may be or encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., heterologous sequences are or encode dystrophin or a functional fragment or variant thereof such as the numerous dystrophin minigenes or microdystrophine coding sequences known in the art), cardiovascular disease (e.g., heterologous sequences are or encode SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., heterologous sequences is or encodes NGF, BDNF, GDNF, NT-3, etc.).

As additional non-limiting example, the heterologous nucleic acid sequence may be or encode an agent that enhances tumor killing activity such as, but not limited to, TRAIL or tumor necrosis factor (TNF), in a cancer. As another non-limiting example, the heterologous nucleic acid sequence may be or encode an agent suitable for the treatment of conditions such as muscular dystrophy (e.g., heterologous nucleic acid sequence is or encodes Dystrophin), cardiovascular disease (e.g., heterologous nucleic acid sequence is or encodes SERCA2a, GATA4, Tbx5, Mef2C, Hand2, Myocd, etc.), neurodegenerative disease (e.g., heterologous nucleic acid sequence is or encodes NGF, BDNF, GDNF, NT-3, etc.), chronic pain (e.g., heterologous nucleic acid sequence is or encodes GlyRal), an enkephalin, or a glutamate decarboxylase (e.g., heterologous nucleic acid sequence is or encodes GAD65, GAD67, or another isoform), lung disease (e.g., heterologous nucleic acid sequence is or encodes CFTR), hemophilia (e.g., heterologous nucleic acid sequence is or encodes Factor VIII or Factor IX), neoplasia (e.g., heterologous nucleic acid sequence is or encodes PTEN, ATM, ATR, EGFR, ERBB2, ERBB3, ERBB4, Notch1, Notch2, Notch3, Notch4, AKT, AKT2, AKT3, HIF, HI F1a, HIF3a, Met, HRG, Bcl2, PPARalpha, PPAR gamma, WT1 (Wilms Tumor), FGF Receptor Family members (5 members: 1, 2, 3, 4, 5), CDKN2a, APC, RB (retinoblastoma), MEN1, VHL, BRCA1, BRCA2, AR (Androgen Receptor), TSG101, IGF, IGF Receptor, Igf1 (4 variants), Igf2 (3 variants), Igf1 Receptor, Igf2 Receptor, Bax, Bcl2, caspases family (9 members: 1, 2, 3, 4, 6, 7, 8, 9, 12), Kras, Ape), age-related macular degeneration (e.g., heterologous nucleic acid sequence is or encodes Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin D, Vldlr), schizophrenia (e.g., Neuregulin (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2 Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HIT (Slc6a4), COMT, DRD (Drdla), SLC6A3, DAOA, DTNBPI, Dao (Dao1)), trinucleotide repeat disorders (e.g., HTT (Huntington's Dx), SBMA/SMAXI/AR (Kennedy's Dx), FXN/X25 (Friedrich's Ataxia), ATX3 (Machado-Joseph's Dx), ATXNI and ATXN2 (spinocerebellar ataxias), DMPK (myotonic dystrophy), Atrophin-1 and Atn1(DRPLA Dx), CBP (Creb-BP-global instability), VLDLR (Alzheimer's), Atxn7, Atxn10), fragile X syndrome (e.g., heterologous nucleic acid sequence is or encodes FMR2, FXRI, FXR2, mGLUR5), secretase related disorders (e.g., heterologous nucleic acid sequence is or encodes APH-1 (alpha and beta), Presenilin (Psen1), nicastrin (Ncstn), PEN-2), ALS (e.g., heterologous nucleic acid sequence is or encodes SOD1, ALS2, STEX, FUS, TARD BP, VEGF (VEGF-a, VEGF-b, VEGF-c)), autism (e.g., heterologous nucleic acid sequence is or encodes Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1), Alzheimer's disease (e.g., heterologous nucleic acid sequence is or encodes E1, CHIP, UCH, UBB, Tau, LRP, PICALM, Clusterin, PS1, SORL1, CR1, Vldlr, Uba1, Uba3, CHIP28 (Aqp1, Aquaporin 1), Uch11, Uch13, APP), inflammation (e.g., heterologous nucleic acid sequence is or encodes IL-10, IL-1 (IL-Ia, IL-Ib), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cll), Parkinson's Disease (e.g., x-Synuclein, DJ-1, LRRK2, Parkin, PINK1), blood and coagulation disorders, such as, e.g., anemia, bare lymphocyte syndrome, bleeding disorders, hemophagocytic lymphohistiocytosis disorders, hemophilia A, hemophilia B, hemorrhagic disorders, leukocyte deficiencies and disorders, sickle cell anemia, and thalassemia (e.g., heterologous nucleic acid sequence is or encodes CRAN1, CDA1, RPS19, DBA, PKLR, PK1, NT5C3, UMPH1, PSNI, RHAG, RH50A, NRAMP2, SPTB, ALAS2, ANH1, ASB, ABCB7, ABC7, ASAT, TAPBP, TPSN, TAP2, ABCB3, PSF2, RING11, MHC2TA, C2TA, RFX5, RFXAP, RFX5, TBXA2R, P2RX1, P2X1, HF1, CFH, HUS, MCFD2, FANCA, FAC A, FA1, FA, FA A, FAAP95, FAAP90, FLJ34064, FANCB, FANCC, FACC, BRCA2, FANCDI, FANCD2, FANCD, FACD, FAD, FANCE, FACE, FANCF, XRCC9, FANCG, BR1PI, BACH1, FANCJ, PHF9, FANCL, FANCM, KIAA1596, PRF1, HPLH2, UNC13D, MUNC13-4, HPLH3, HLH3, FHL3, F8, FSC, PI, ATT, F5, ITGB2, CD18, LCAMB, LAD, EIF2B1, EIF2BA, EIF2B2, EIF2B3, EIF2B5, LVWM, CACH, CLE, EIF2B4, HBB, HBA2, HBB, HBD, LCRB, HBA1), B-cell non-Hodgkin lymphoma or leukemia (e.g., heterologous nucleic acid sequence is or encodes BCL7A, BCL7, ALI, TCL5, SCL, TAL2, FLT3, NBS1, NBS, ZNFN1AI, 1KI, LYF1, HOXD4, HOX4B, BCR, CML, PHL, ALL, ARNT, KRAS2, RASK2, GMPS, AFIO, ARHGEF12, LARG, KIAA0382, CALM, CLTH, CEBPA, CEBP, CHIC2, BTL, FLT3, KIT, PBT, LPP, NPMI, NUP214, D9S46E, CAN, CAIN, RUNXI, CBFA2, AML1, WHSC1LI, NSD3, FLT3, AF1Q, NPMI, NUMA1, ZNF145, PLZF, PML, MYL, STAT5B, AF1Q, CALM, CLTH, ARL11, ARLTS1, P2RX7, P2X7, BCR, CML, PHL, ALL, GRAF, NF1, VRNF, WSS, NFNS, PTPNII, PTP2C, SHP2, NS1, BCL2, CCND1, PRAD1, BCL1, TCRA, GATA1, GF1, ERYF1, NFE1, ABLI, NQO1, DIA4, NMOR1, NUP214, D9S46E, CAN, CAIN), inflammation and immune related diseases and disorders (e.g., heterologous nucleic acid sequence is or encodes KIR3DL1, NKAT3, NKB1, AMB 11, K1R3DS1, IFNG, CXCL12, TNFRSF6, APT1, FAS, CD95, ALPS1A, IL2RG, SCIDX1, SCIDX, IMD4, CCL5, SCYA5, D17S136E, TCP228, IL10, CSIF, CMKBR2, CCR2, CMKBR5, CCCKR5 (CCR5), CD3E, CD3G, AICDA, AID, HIGM2, TNFRSF5, CD40, UNG, DGU, HIGM4, TNFSFS, CD40LG, HIGM1, IGM, FOXP3, IPEX, AIID, XPID, PIDX, TNFRSF14B, TACI), inflammation (e.g., heterologous nucleic acid sequence is or encodes IL-10, IL-1 (IL-IA, IL-IB), IL-13, IL-17 (IL-17a (CTLA8), IL-17b, IL-17c, IL-17d, IL-171), 11-23, Cx3crl, ptpn22, TNFa, NOD2/CARD15 for IBD, IL-6, IL-12 (IL-12a, IL-12b), CTLA4, Cx3cII), JAK3, JAKL, DCLREIC, ARTEMIS, SCIDA, RAG1, RAG2, ADA, PTPRC, CD45, LCA, IL7R, CD3D, T3D, IL2RG, SCIDXI, SCIDX, IMD4), metabolic, liver, kidney and protein diseases and disorders (e.g., heterologous nucleic acid sequence is or encodes TTR, PALB, APOA1, APP, AAA, CVAP, ADI, GSN, FGA, LYZ, TTR, PALB, KRT18, KRT8, CIRHIA, NAIC, TEX292, KIAA1988, CFTR, ABCC7, CF, MRP7, SLC2A2, GLUT2, G6PC, G6PT, G6PT1, GAA, LAMP2, LAMPB, AGL, GDE, GBE1, GYS2, PYGL, PFKM, TCF1, HNF1A, MODY3, SCOD1, SCO1, CTNNB1, PDGFRL, PDGRL, PRLTS, AX1NI, AXIN, CTNNB1, TP53, P53, LFS1, IGF2R, MPRI, MET, CASP8, MCH5, UMOD, HNFJ, FJHN, MCKD2, ADMCKD2, PAH, PKU1, QDPR, DHPR, PTS, FCYT, PKHD1, ARPKD, PKD1, PKD2, PKD4, PKDTS, PRKCSH, G19P1, PCLD, SEC63), muscular/skeletal diseases and disorders (e.g., heterologous nucleic acid sequence is or encodes DMD, BMD, MYF6, LMNA, LMN1, EMD2, FPLD, CMDIA, HGPS, LGMDIB, LMNA, LMNI, EMD2, FPLD, CMDIA, FSHMD1A, FSHD1A, FKRP, MDC1C, LGMD2I, LAMA2, LAMM, LARGE, KIAA0609, MDC1D, FCMD, TTID, MYOT, CAPN3, CANP3, DYSF, LGMD2B, SGCG, LGMD2C, DMDA1, SCG3, SGCA, ADL, DAG2, LGMD2D, DMDA2, SGCB, LGMD2E, SGCD, SGD, LGMD2F, CMD1L, TCAP, LGMD2G, CMD1N, TRIM32, HT2A, LGMD2H, FKRP, MDCIC, LGMD21, TTN, CMD1G, TMD, LGMD2J, POMT1, CAV3, LGMD1C, SEPN1, SELN, RSMD1, PLEC1, PLTN, EBS1, LRP5, BMND1, LRP7, LR3, OPPG, VBCH2, CLCN7, CLC7, OPTA2, OSTMI, GL, TCIRG1, TIRC7, OC116, OPTB1, VAPB, VAPC, ALS8, SMN1, SMA1, SMA2, SMA3, SMA4, BSCL2, SPG17, GARS, SMAD1, CMT2D, HEXB, IGHMBP2, SMUBP2, CATF1, SMARD1), neurological and neuronal diseases and disorders (e.g., heterologous nucleic acid sequence is or encodes SOD1, ALS2, STEX, FUS, TARDBP, VEGF (VEGF-a, VEGF-b, VEGF-c), APP, AAA, CVAP, ADI, APOE, AD2, PSEN2, AD4, STM2, APBB2, FE65LI, NOS3, PLAU, URK, ACE, DCPI, ACEI, MPO, PAC1PI, PAXIPIL, PTIP, A2M, BLMH, BMH, PSEN1, AD3, Mecp2, BZRAP1, MDGA2, Sema5A, Neurexin 1, GLO1, MECP2, RTT, PPMX, MRX16, MRX79, NLGN3, NLGN4, KIAA1260, AUTSX2, FMR2, FXR1, FXR2, mGLUR5, HD, IT15, PRNP, PRIP, JPH3, JP3, HDL2, TBP, SCA17, NR4A2, NURR1, NOT, TINUR, SNCAIP, TBP, SCA17, SNCA, NACP, PARK1, PARK4, DJI, PARK7, LRRK2, PARK8, PINK1, PARK6, UCHL1, PARK5, SNCA, NACP, PARK1, PARK4, PRKN, PARK2, PDJ, DBH, NDUFV2, MECP2, RTT, PPMX, MRX16, MRX79, CDKL5, STK9, MECP2, RTT, PPMX, MRX16, MRX79, x-Synuclein, DJ-1, Neuregulin-1 (Nrgl), Erb4 (receptor for Neuregulin), Complexin-1 (Cplx1), Tph1 Tryptophan hydroxylase, Tph2, Tryptophan hydroxylase 2, Neurexin 1, GSK3, GSK3a, GSK3b, 5-HTT (Slc6a4), CONT, DRD (Drdla), SLC6A, DAOA, DTNBP1, Dao (Daol), APH-1(alpha and beta), Presenilin (Psenl), Nicastrin, (Ncstn), PEN-2, Nos1, Parp1, Nat1, Nat2, HTT, SBMA/SMAX1/AR, FXN/X25, ATX3, TXN, ATXN2, DMPK, Atrophin-1, Atn1, CBP, VLDLR, Atxn7, and AtxnlO), and ocular diseases and disorders (e.g., Aber, Ccl2, Cc2, cp (ceruloplasmin), Timp3, cathepsin-D, Vldlr, Ccr2, CRYAA, CRYA1, CRYBB2, CRYB2, PITX3, BFSP2, CP49, CP47, CRYAA, CRYAI, PAX6, AN2, MGDA, CRYBA1, CRYB1, CRYGC, CRYG3, CCL, LIM2, MP19, CRYGD, CRYG4, BFSP2, CP49, CP47, HSF4, CTM, HSF4, CTM, MIP, AQPO, CRYAB, CRYA2, CTPP2, CRYBB1, CRYGD, CRYG4, CRYBB2, CRYB2, CRYGC, CRYG3, CCL, CRYAA, CRYAI, GJA8, CX50, CAE1, GJA3, CX46, CZP3, CAE3, CCM1, CAM, KRIT1, APOA1, TGFBI, CSD2, CDGG1, CSD, BIGH3, CDG2, TACSTD2, TROP2, M1SI, VSX1, RINX, PPCD, PPD, KTCN, COL8A2, FECD, PPCD2, PIP5K3, CFD, KERA, CNA2, MYOC, TIGR, GLCIA, JO AG, GPOA, OPTN, GLC1E, FIP2, HYPL, NRP, CYP1BI, GLC3A, OPA1, NTG, NPG, CYP1BI, GLC3A, CRB1, RP12, CRX, CORD2, CRD, RPGRIPI, LCA6, CORD9, RPE65, RP20, AIPL1, LCA4, GUCY2D, GUC2D, LCA1, CORD6, RDH12, LCA3, ELOVL4, ADMD, STGD2, STGD3, RDS, RP7, PRPH2, PRPH, AVMD, AOFMD, and VMD2).

In some embodiments, the heterologous nucleic acid sequence is or encodes a factor that can affect the differentiation of a cell. As a non-limiting example, the expression of one or more of Oct4, Klf4, Sox2, c-Myc, L-Myc, dominant-negative p53, Nanog, Glis1, Lin28, TFIID, mir-302/367, or other miRNAs can cause the cell to become an induced pluripotent stem (iPS) cell.

In some embodiments, the heterologous nucleic acid sequence is or encodes a factor for transdifferentiating cells. Non-limiting examples of factors include: one or more of GATA4, Tbx5, Mef2C, Myocd, Hand2, SRF, Mesp1, SMARCD3 for cardiomyocytes; Ascii, Nurr1, Lmx1A, Bm2, Myt11, NeuroD1, FoxA2 for neural cells; and Hnf4a, Foxa1, Foxa2 or Foxa3 for hepatic cells.

In certain embodiments, the heterologous nucleic acid encodes a therapeutic antibody, or an antigen-binding fragment thereof.

In certain embodiments, the heterologous nucleic acid encodes a protein for replacement therapy. The protein may be defective in a disease cell or a disease organism/individual, and the wild-type protein or a functional fragment or variant thereof, when delivered by the heterologous nucleic acid to the disease cell/tissue/organism/individual, at least partially or fully restores the lost function of the protein the disease cell/tissue/organism/individual.

HNS=Donor DNA Template

In some embodiments, the heterologous nucleic acid sequence is a donor DNA template that can be integrated into a host genome via HDR.

In some embodiments, the heterologous nucleic acid sequence is a donor DNA that can serve as a template or primer for recombineering during replication.

In certain embodiments, the heterologous nucleic acid comprises or encodes a donor/template sequence, wherein the donor/template corrects/repairs/removes a mutation at the target genome site. For example, the mutation may be a mutated exon in a disease gene.

In certain embodiments, the donor/template may encode or comprises a functional DNA element, such as a promoter, an enhancer, a protein binding sequence, a methylation site, or a homology region for assisting gene editing, etc.

By “donor DNA” or “donor DNA template” it is meant a single-stranded DNA to be inserted at a site cleaved by a gene-editing nuclease (e.g., a CRISPR/Cas effector protein; a TALEN; a ZFN) (e.g., after dsDNA cleavage, after nicking a target DNA, after dual nicking a target DNA, and the like). The donor DNA template can contain sufficient homology to a genomic sequence at the target site, e.g., 70%, 80%, 85%, 90%, 95%, or 100% homology with the nucleotide sequences flanking the target site, e.g. within about 50 bases or less of the target site, e.g. within about 30 bases, within about 15 bases, within about 10 bases, within about 5 bases, or immediately flanking the target site, to support homology-directed repair between it and the genomic sequence to which it bears homology.

Approximately 25, 50, 100, or 200 nucleotides, or more than 200 nucleotides, of sequence homology between a donor DNA template and a genomic sequence (or any integral value between 10 and 200 nucleotides, or more) can support homology-directed repair. Donor DNA template can be of any length, e.g., 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 500 nucleotides or more, 1000 nucleotides or more, 5000 nucleotides or more, etc. A suitable donor DNA template can be from 50 nucleotides to 100 nucleotides, from 100 nucleotides to 500 nucleotides, from 500 nucleotides to 1000 nucleotides, from 1000 nucleotides to 5000 nucleotides, or from 5000 nucleotides to 10,000 nucleotides, or more than 10,000 nucleotides, in length.

As noted above, the donor DNA template comprises a first homology arm and a second homology arm. The first homology arm is at or near the 5′ end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to a first nucleotide sequence in a target nucleic acid. The second homology arm is at or near the 3′ end of the donor DNA; and comprises a nucleotide sequence that is at least partially complementary to a second nucleotide sequence in the target nucleic acid. The first and second homology arms can each independently have a length of from about 10 nucleotides to 400 nucleotides; e.g., from 10 nucleotides (nt) to 15 nt, from 15 nt to 20 nt, from 20 nt to 25 nt, from 25 nt to 30 nt, from 30 nt to 35 nt, from 35 nt to 40 nt, from 40 nt to 45 nt, from 45 nt to 50 nt, from 50 nt to 75 nt, from 75 nt to 100 nt, from 100 nt to 125 nt, from 125 nt to 150 nt, from 150 nt to 175 nt, from 175 nt to 200 nt, from 200 nt to 225 nt, from 225 nt to 250 nt, from 250 nt to 275 nt, from 275 nt to 300 nt, from 325 nt to 350 nt, from 350 nt to 375 nt, or from 375 nt to 400 nt.

In certain embodiments, the donor DNA template is used for editing the target nucleotide sequence. In certain embodiments, the donor DNA template comprises one or more mutations to be introduced into the target polynucleotide. Examples of such mutations include substitutions, deletions, insertions, or a combination thereof. In certain embodiments, the mutation causes a shift in an open reading frame on the target polynucleotide. In certain embodiments, the donor polynucleotide alters a stop codon in the target polynucleotide. In certain embodiments, the donor polynucleotide corrects a premature stop codon. The correction can be achieved by deleting the stop codon, or by introducing one or more sequence changes to alter the stop codon to a codon. In certain embodiments, the donor polynucleotide addresses loss of function mutations, deletions, or translocations that may occur, for example, in certain disease contexts by inserting or restoring a functional copy of a gene, or functional fragment thereof, or a functional regulatory sequence or functional fragment of a regulatory sequence. A functional fragment includes a fragment less than the entire copy of a gene but otherwise provides sufficient nucleotide sequence to restore the functionality of a wild type gene or non-coding regulatory sequence (e.g., sequences encoding long non-coding RNA).

In certain embodiments, the donor DNA template may be used to replace a single allele of a defective gene or defective fragment thereof. In another embodiment, the donor DNA template is used to replace both alleles of a defective gene or defective gene fragment. A “defective gene” or “defective gene fragment” is a gene or portion of a gene that when expressed, fails to generate a functioning protein or non-coding RNA with functionality of the corresponding wild-type gene.

In certain example embodiments, these defective genes may be associated with one or more disease phenotypes. In certain example embodiments, the defective gene or gene fragment is not replaced but the heterologous nucleic acid is used to insert donor polynucleotides that encode gene or gene fragments that compensate for or override defective gene expression such that cell phenotypes associated with defective gene expression are eliminated or changed to a different or desired cellular phenotype. This can be achieved by including the coding sequence of a therapeutic protein, such as a therapeutic antibody or functional fragment thereof, or a wild-type version of a defective protein associated with one or more disease phenotypes.

In certain embodiments, the donor may include, but not be limited to, genes or gene fragments, encoding proteins or RNA transcripts to be expressed, regulatory elements, repair templates, and the like. According to the invention, the donor polynucleotides may comprise left end and right end sequence elements that function with transposition components that mediate insertion.

In certain embodiments, the donor DNA template manipulates a splicing site on the target polynucleotide. In certain embodiments, the donor DNA template disrupts a splicing site. The disruption may be achieved by inserting the polynucleotide to a splicing site and/or introducing one or more mutations to the splicing site. In certain embodiments, the donor polynucleotide may restore a splicing site. For example, the polynucleotide may comprise a splicing site sequence.

In certain embodiments, the donor DNA template to be inserted has a size from 10 bp to 50 kb in length, e.g., from 50 bp to ˜40 kb, from 100 bp to ˜30 kb, from 100 bp to ˜10 kb, from 100 bp to 300 bp, from 200 bp to 400 bp, from 300 bp to 500 bp, from 400 bp to 600 bp, from 500 bp to 700 bp, from 600 bp to 800 bp, from 700 bp to 900 bp, from 800 bp to 1000 bp, from 900 bp to 1100 bp, from 1000 bp to 1200 bp, from 1100 bp to 1300 bp, from 1200 bp to 1400 bp, from 1300 bp to 1500 bp, from 1400 bp to 1600 bp, from 1500 bp to 1700 bp, from 1600 bp to 1800 bp, from 1700 bp to 1900 bp, from 1800 bp to 2000 bp nucleotides in length.

In certain embodiments, the homologous arm on one or both ends of the sequence to be inserted is independently about 20 bp, 40 bp, 60 bp, 80 bp, 100 bp, 120 bp, or 150 bp.

The first homology arm and the second homology arm of the donor DNA flank a nucleotide sequence (“a nucleotide sequence of interest” or “an intervening nucleotide sequence”) that is to be introduced into a target nucleic acid. The nucleotide sequence of interest can comprise: i) a nucleotide sequence encoding a polypeptide of interest; ii) a nucleotide sequence encoding an exon of a gene; iii) a promoter sequence; iv) an enhancer sequence; v) a nucleotide sequence encoding a non-coding RNA; or vi) any combination of the foregoing.

The donor DNA can provide for gene correction, gene replacement, gene tagging, transgene insertion, nucleotide deletion, gene disruption, gene mutation, etc. For example, the donor DNA can be used to add, e.g., insert or replace, nucleic acid material to a target DNA (e.g. to “knock in” a nucleic acid that encodes a protein, an siRNA, an miRNA, etc.), to add a tag (e.g., 6×His, a fluorescent protein (e.g., a green fluorescent protein; a yellow fluorescent protein, etc.), hemagglutinin (HA), FLAG, etc.), to add a regulatory sequence to a gene (e.g. promoter, polyadenylation signal, internal ribosome entry sequence (IRES), 2A peptide, start codon, stop codon, splice signal, localization signal, enhancer, etc.), to modify a nucleic acid sequence (e.g., introduce a mutation), and the like. For example, the donor DNA can be used to modify DNA in a site-specific, i.e. “targeted”, way; for example gene knock-out, gene knock-in, gene editing, gene tagging, etc., as used in, for example, gene therapy, e.g. to treat a disease; or as an antiviral, antipathogenic, or anticancer therapeutic, the production of genetically modified organisms in agriculture, the large scale production of proteins by cells for therapeutic, diagnostic, or research purposes, the induction of pluripotent stem cells, biological research, the targeting of genes of pathogens for deletion or replacement, etc.

In some cases, the donor DNA comprises a nucleotide sequence encoding a polypeptide of interest. Polypeptides of interest include, e.g., a) functional versions of a polypeptide that comprises one or more amino acid substitutions, insertions, and/or deletions and that exhibits reduced function, e.g., where the reduced function is associated with or causes a pathological condition; b) fluorescent polypeptides; c) hormones; d) receptors for ligands; e) ion channels; f) neurotransmitters; g) and the like.

In some cases, the donor DNA comprises a nucleotide sequence that encodes a wild-type protein that is lacking in the recipient cell. In some cases, the donor DNA encodes a wild type factor (e.g. Factor VII, Factor VIII, Factor IX and the like) involved in coagulation. In some cases, the donor DNA comprises a nucleotide sequence that encodes a therapeutic antibody. In some cases, the donor DNA comprises a nucleotide sequence that encodes an engineered protein or receptor. In some cases, the engineered receptor is a T cell receptor (TCR), a natural killer (NK) receptor (NKR), or a B cell receptor (BCR). In some cases, the engineered TCR or NKR targets a cancer marker (e.g., a polypeptide that is expressed (e.g., over-expressed) on the surface of a cancer cell). In some cases, the donor DNA comprises a nucleotide sequence that encodes a chimeric antigen receptor (CAR). In some cases, the CAR targets a cancer marker. Donor DNAs encoding CAR, TCR, and/or NCR proteins may be folded into DNA origami structures (DNA nanostructures) and delivered into T cells or NK cells in vitro or in vivo.

Non-limiting examples of polypeptides that can be encoded by a donor DNA include, e.g., IL1B (interleukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTGIS (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin), IL4 (interleukin 4), ANGPT1 (angiopoietin 1), ABCG8 (ATP-binding cassette, sub-family G (WHITE), member 8), CTSK (cathepsin K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)), KCNJ11 (potassium inwardly-rectifying channel, subfamily J, member 11), INS (insulin), CRP (C-reactive protein, pentra)(in-related), PDGFRB (platelet-derived growth factor receptor, beta polypeptide), CCNA2 (cyclin A2), PDGFB (platelet-derived growth factor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)), KCNJ5 (potassium inwardly-rectifying channel, subfamily J, member 5), KCNN3 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 3), CAPN10 (calpain 10), PTGES (prostaglandin E synthase), ADRA2B (adrenergic, alpha-2B-, receptor), ABCG5 (ATP-binding cassette, sub-family G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAPN5 (calpain 5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C (mex-3 homolog C (C. elegans)), ACE angiotensin I converting enzyme (peptidyl-dipeptidase A) 1), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6 (interleukin 6 (interferon, beta 2)), STN (statin), SERPINE1 (serpin peptidase inhibitor, clade E (nexin, plasminogen activator inhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin, C1Q and collagen domain containing), APOB (apolipoprotein B (including Ag(x) antigen)), APOE (apolipoprotein E), LEP (leptin), MTHFR (5,10-methylenetetrahydrofolate reductase (NADPH)), APOA1 (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (natriuretic peptide precursor B), NOS3 (nitric oxide synthase 3 (endothelial cell)), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (plasminogen activator, tissue), PTGS2 (prostaglandin-endoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)), CETP (cholesteryl ester transfer protein, plasma), AGTR1 (angiotensin II receptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme A reductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE (selectin E), REN (renin), PPARA (peroxisome proliferator-activated receptor alpha), PON1 (paraoxonase 1), KNG1 (kininogen 1), CCL2 (chemokine (C-C motif) ligand 2), LPL (lipoprotein lipase), vWF (von Willebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transforming growth factor, beta 1), NPPA (natriuretic peptide precursor A), IL10 (interleukin 10), EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM1 (vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA (lipoprotein, Lp(a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1), MAPK1 (mitogen-activated protein kinase 1), HP (haptoglobin), F3 (coagulation factor III (thromboplastin, tissue factor)), CST3 (cystatin C), COG2 (component of oligomeric Golgi complex 2), MMP9 (matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase)), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VIII, procoagulant component), HMOX1 (heme oxygenase (decycling) 1), APOC3 (apolipoprotein C-III), IL8 (interleukin 8), PROK1 (prokineticin 1), CBS (cystathionine-beta-synthase), NOS2 (nitric oxide synthase 2, inducible), TLR4 (toll-like receptor 4), SELP (selectin P (granule membrane protein 140 kDa, antigen CD62)), ABCA1 (ATP-binding cassette, sub-family A (ABC1), member 1), AGT (angiotensinogen (serpin peptidase inhibitor, clade A, member 8)), LDLR (low density lipoprotein receptor), GPT (glutamic-pyruvate transaminase (alanine aminotransferase)), VEGFA (vascular endothelial growth factor A), NR3C2 (nuclear receptor subfamily 3, group C, member 2), IL18 (interleukin 18 (interferon-gamma-inducing factor)), NOS1 (nitric oxide synthase 1 (neuronal)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrinogen beta chain), HGF (hepatocyte growth factor (hepapoietin A; scatter factor)), ILIA (interleukin 1, alpha), RETN (resistin), AKT1 (v-akt murine thymoma viral oncogene homolog 1), LIPC (lipase, hepatic), HSPD1 (heat shock 60 kDa protein 1 (chaperonin)), MAPK14 (mitogen-activated protein kinase 14), SPP1 (secreted phosphoprotein 1), ITGB3 (integrin, beta 3 (platelet glycoprotein 111a, antigen CD61)), CAT (catalase), UTS2 (urotensin 2), THBD (thrombomodulin), F10 (coagulation factor X), CP (ceruloplasmin (ferroxidase)), TNFRSF11B (tumor necrosis factor receptor superfamily, member lib), EDNRA (endothelin receptor type A), EGFR (epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)), MMP2 (matrix metallopeptidase 2 (gelatinase A, 72 kDa gelatinase, 72 kDa type IV collagenase)), PLG (plasminogen), NPY (neuropeptide Y), RHOD (ras homolog gene family, member D), MAPK8 (mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis viral oncogene homolog (avian)), FN1 (fibronectin 1), CMA1 (chymase 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide binding protein (G protein), beta polypeptide 3), ADRB2 (adrenergic, beta-2-, receptor, surface), APOA5 (apolipoprotein A-V), SOD2 (superoxide dismutase 2, mitochondrial), F5 (coagulation factor V (proaccelerin, labile factor)), VDR (vitamin D (1,25-dihydroxyvitamin D3) receptor), ALOX5 (arachidonate 5-lipoxygenase), HLA-DRB1 (major histocompatibility complex, class II, DR beta 1), PARP1 (poly (ADP-ribose) polymerase 1), CD40LG (CD40 ligand), PON2 (paraoxonase 2), AGER (advanced glycosylation end product-specific receptor), IRS1 (insulin receptor substrate 1), PTGS1 (prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase)), ECE1 (endothelin converting enzyme 1), F7 (coagulation factor VII (serum prothrombin conversion accelerator)), URN (interleukin 1 receptor antagonist), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1 (insulin-like growth factor binding protein 1), MAPK10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily, member 6)), ABCB1 (ATP-binding cassette, sub-family B (MDR/TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growth factor binding protein 3), CD14 (CD14 molecule), PDE5A (phosphodiesterase 5A, cGMP-specific), AGTR2 (angiotensin II receptor, type 2), CD40 (CD40 molecule, TNF receptor superfamily member 5), LCAT (lecithin-cholesterol acyltransferase), CCR5 (chemokine (C-C motif) receptor 5), MMP1 (matrix metallopeptidase 1 (interstitial collagenase)), TIMP1 (TIMP metallopeptidase inhibitor 1), ADM (adrenomedullin), DYT10 (dystonia 10), STAT3 (signal transducer and activator of transcription 3 (acute-phase response factor)), MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatinase)), ELN (elastin), USF1 (upstream transcription factor 1), CFH (complement factor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), MME (membrane metallo-endopeptidase), F2R (coagulation factor II (thrombin) receptor), SELL (selectin L), CTSB (cathepsin B), ANXA5 (annexin A5), ADRB1 (adrenergic, beta-1-, receptor), CYBA (cytochrome b-245, alpha polypeptide), FGA (fibrinogen alpha chain), GGT1 (gamma-glutamyltransferase 1), LIPG (lipase, endothelial), HIF1A (hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)), CXCR4 (chemokine (C-X-C motif) receptor 4), PROC (protein C (inactivator of coagulation factors Va and Villa)), SCARB1 (scavenger receptor class B, member 1), CD79A (CD79a molecule, immunoglobulin-associated alpha), PLTP (phospholipid transfer protein), ADD1 (adducin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid A1), KCNH2 (potassium voltage-gated channel, subfamily H (eag-related), member 2), DPP4 (dipeptidyl-peptidase 4), G6PD (glucose-6-phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A)), VTN (vitronectin), KIAA0101 (KIAA0101), FOS (FBJ murine osteosarcoma viral oncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolyl isomerase G (cyclophilin G)), ILIR1 (interleukin 1 receptor, type I), AR (androgen receptor), CYP1A1 (cytochrome P450, family 1, subfamily A, polypeptide 1), SERPINA1 (serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1), MTR (5-methyltetrahydrofolate-homocysteine methyltransferase), RBP4 (retinol binding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptor type B), ITGA2 (integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)), CAB INI (calcineurin binding protein 1), SHBG (sex hormone-binding globulin), HMGB1 (high-mobility group box 1), HSP90B2P (heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4), GJA1 (gap junction protein, alpha 1, 43 kDa), CAV1 (caveolin 1, caveolae protein, 22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha (TNF superfamily, member 1)), GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (beta polypeptide)), SP1 (Sp 1 transcription factor), TGIF1 (TGFB-induced factor homeobox 1), SRC (v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian)), EGF (epidermal growth factor (beta-urogastrone)), PIK3CG (phosphoinositide-3-kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibility complex, class I, A), KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrillin 1), CHKA (choline kinase alpha), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursor protein), CTNNB1 (catenin (cadherin-associated protein), beta 1, 88 kDa), IL2 (interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, beta 1 non-catalytic subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7 family, member A1), CX3CR1 (chemokine (C-X3-C motif) receptor 1), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GH1 (growth hormone 1), TF (transferrin), HFE (hemochromatosis), IE17A (interleukin 17A), PTEN (phosphatase and tensin homolog), GSTM1 (glutathione S-transferase mu 1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1 (coagulation factor XIII, A1 polypeptide), TTR (transthyretin), FABP4 (fatty acid binding protein 4, adipocyte), PON3 (paraoxonase 3), APOC1 (apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor superfamily, member IB), HTR2A (5-hydroxytryptamine (serotonin) receptor 2A), CSF3 (colony stimulating factor 3 (granulocyte)), CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9), TXN (thioredoxin), CYP11B2 (cytochrome P450, family 11, subfamily B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colony stimulating factor 2 (granulocyte-macrophage)), KDR (kinase insert domain receptor (a type III receptor tyrosine kinase)), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid)), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCG (glucagon), RHOA (ras homolog gene family, member A), ALDH2 (aldehyde dehydrogenase 2 family (mitochondrial)), TCF7L2 (transcription factor 7-like 2 (T-cell specific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclear factor (erythroid-derived 2)-like 2), NOTCH1 (Notch homolog 1, translocation-associated (Drosophila)), UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1), IFNA1 (interferon, alpha 1), PPARD (peroxisome proliferator-activated receptor delta), SIRT1 (sirtuin (silent mating type information regulation 2 homolog) 1 (S. cerevisiae)), GNRH1 (gonadotropin-releasing hormone 1 (luteinizing-releasing hormone)), PAPPA (pregnancy-associated plasma protein A, pappalysin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC (natriuretic peptide precursor C), AHSP (alpha hemoglobin stabilizing protein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13), MTOR (mechanistic target of rapamycin (serine/threonine kinase)), ITGB2 (integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)), GSTT1 (glutathione S-transferase theta 1), IL6ST (interleukin 6 signal transducer (gp130, oncostatin M receptor)), CPB2 (carboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrier family 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosolic, calcium-independent)), TNFSF11 (tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solute carrier family 8 (sodium/calcium exchanger), member 1), F2RL1 (coagulation factor II (thrombin) receptor-like 1), AKR1A1 (aldo-keto reductase family 1, member A1 (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase 9 family, member A1), BGLAP (bone gamma-carboxyglutamate (gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR (5-methyltetrahydrofolate-homocysteine methyltransferase reductase), SULT1A3 (sulfotransferase family, cytosolic, IA, phenol—preferring, member 3), RAGE (renal tumor antigen), C4B (complement component 4B (Chido blood group), P2RY12 (purinergic receptor P2Y, G-protein coupled, 12), RNLS (renalase, FAD-dependent amine oxidase), CREB1 (cAMP responsive element binding protein 1), POMC (proopiomelanocortin), RAC1 (ras-related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Racl)), LMNA (lamin NC), CD59 (CD59 molecule, complement regulatory protein), SCN5A (sodium channel, voltage-gated, type V, alpha subunit), CYP1B1 (cytochrome P450, family 1, subfamily B, polypeptide 1), MIF (macrophage migration inhibitory factor (glycosylation-inhibiting factor)), MMP13 (matrix metallopeptidase 13 (collagenase 3)), TIMP2 (TIMP metallopeptidase inhibitor 2), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1), CYP21A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor type 22 (lymphoid)), MYH14 (myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonic defect)), SELPLG (selectin P ligand), AOC3 (amine oxidase, copper containing 3 (vascular adhesion protein 1)), CTSL1 (cathepsin LI), PCNA (proliferating cell nuclear antigen), IGF2 (insulin like growth factor 2 (somatomedin A)), ITGB1 (integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12)), CAST (calpastatin), CXCL12 (chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)), IGHE (immunoglobulin heavy constant epsilon), KCNE1 (potassium voltage-gated channel, Isk-related family, member 1), TFRC (transferrin receptor (p90, CD71)), COL1A1 (collagen, type I, alpha 1), COL1A2 (collagen, type I, alpha 2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTPN11 (protein tyrosine phosphatase, non-receptor type 11), SLC2A1 (solute carrier family 2 (facilitated glucose transporter), member 1), IL2RA (interleukin 2 receptor, alpha), CCL5 (chemokine (C-C motif) ligand 5), IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-like apoptosis regulator), CALC A (calcitonin-related polypeptide alpha), EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (glutathione S-transferase pi 1), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (heparan sulfate proteoglycan 2), CCL3 (chemokine (C-C motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRBK1 (adrenergic, beta, receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member 2), MMP8 (matrix metallopeptidase 8 (neutrophil collagenase)), NPR2 (natriuretic peptide receptor B/guanylate cyclase B (atrionatriuretic peptide receptor B)), GCH1 (GTP cyclohydrolase 1), EPRS (glutamyl-prolyl-tRNA synthetase), PPARGC1A (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), F12 (coagulation factor XII (Hageman factor)), PEC AMI (platelet/endothelial cell adhesion molecule), CCL4 (chemokine (C-C motif) ligand 4), SERPINA3 (serpin peptidase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3), CASR (calcium-sensing receptor), GJA5 (gap junction protein, alpha 5, 40 kDa), FABP2 (fatty acid binding protein 2, intestinal), TTF2 (transcription termination factor, RNA polymerase II), PROS1 (protein S (alpha)), CTF1 (cardiotrophin 1), SGCB (sarcoglycan, beta (43 kDa dystrophin-associated glycoprotein)), YME1L1 (YME1-like 1 (S. cerevisiae)), CAMP (cathelicidin antimicrobial peptide), ZC3H12A (zinc finger CCCH-type containing 12A), AKR1B1 (aldo-keto reductase family 1, member B1 (aldose reductase)), DES (desmin), MMP7 (matrix metallopeptidase 7 (matrilysin, uterine)), AHR (aryl hydrocarbon receptor), CSF1 (colony stimulating factor 1 (macrophage)), HDAC9 (histone deacetylase 9), CTGF (connective tissue growth factor), KCNMA1 (potassium large conductance calcium-activated channel, subfamily M, alpha member 1), UGT1A (UDP glucuronosyltransferase 1 family, polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT (catechol-b-methyltransferase), S100B (S100 calcium binding protein B), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine receptor D4), CAMK2G (calcium/calmodulin-dependent protein kinase II gamma), SLC22A2 (solute carrier family 22 (organic cation transporter), member 2), CCL11 (chemokine (C-C motif) ligand 11), PGF (placental growth factor), THPO (thrombopoietin), GP6 (glycoprotein VI (platelet)), TACR1 (tachykinin receptor 1), NTS (neurotensin), HNF1A (HNF1 homeobox A), SST (somatostatin), KCND1 (potassium voltage-gated channel, Shal-related subfamily, member 1), LOC646627 (phospholipase inhibitor), TBXAS1 (thromboxane A synthase 1 (platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH1C (alcohol dehydrogenase 1C (class I), gamma polypeptide), ALOX12 (arachidonate 12-lipoxygenase), AHSG (alpha-2-HS-glycoprotein), BHMT (betaine-homocysteine methyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa), SLC25A4 (solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonate 5-lipoxygenase-activating protein), NUMA1 (nuclear mitotic apparatus protein 1), CYP27B1 (cytochrome P450, family 27, subfamily B, polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SOD3 (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GHRL (ghrelin/obestatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBD10 (kelch repeat and BTB (POZ) domain containing 10), TNC (tenascin C), TYMS (thymidylate synthetase), SHC1 (SHC (Src homology 2 domain containing) transforming protein 1), LRP1 (low density lipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokine signaling 3), ADH1B (alcohol dehydrogenase IB (class I), beta polypeptide), KLK3 (kallikrein-related peptidase 3), HSD11B1 (hydroxysteroid (11-beta) dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subunit 1), SERPINB2 (serpin peptidase inhibitor, clade B (ovalbumin), member 2), TNS1 (tensin 1), RNF19A (ring finger protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M (complement component 3 receptor 3 subunit)), PITX2 (paired-like homeodomain 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fc fragment of IgG, low affinity 111a, receptor (CD16a)), LEPR (leptin receptor), ENG (endoglin), GPX1 (glutathione peroxidase 1), GOT2 (glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2)), HRH1 (histamine receptor HI), NR112 (nuclear receptor subfamily 1, group I, member 2), CRH (corticotropin releasing hormone), HTR1A (5-hydroxytryptamine (serotonin) receptor 1A), VDAC1 (voltage-dependent anion channel 1), HPSE (heparanase), SFTPD (surfactant protein D), TAP2 (transporter 2, ATP-binding cassette, sub-family B (MDR/TAP)), RNF123 (ring finger protein 123), PTK2B (PTK2B protein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase, receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQOl (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor subfamily 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26 kDa), SLC9A1 (solute carrier family 9 (sodium/hydrogen exchanger), member 1), MAOA (monoamine oxidase A), PCSK9 (proprotein convertase subtilisin/kexin type 9), FCGR2A (Fc fragment of IgG, low affinity Ila, receptor (CD32)), SERPINF1 (serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1), EDN3 (endothelin 3), DHFR (dihydrofolate reductase), GAS6 (growth arrest-specific 6), SMPD1 (sphingomyelin phosphodiesterase 1, acid lysosomal), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)), TFAP2A (transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)), C4BPA (complement component 4 binding protein, alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Regan isozyme)), CXCR2 (chemokine (C-X-C motif) receptor 2), SLC39A3 (solute carrier family 39 (zinc transporter), member 3), ABCG2 (ATP-binding cassette, sub-family G (WHITE), member 2), ADA (adenosine deaminase), JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein IA), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), Fll (coagulation factor XI), ATP7A (ATPase, Cu++ transporting, alpha polypeptide), CR1 (complement component (3b/4b) receptor 1 (Knops blood group)), GFAP (glial fibrillary acidic protein), ROCK1 (Rho-associated, coiled-coil containing protein kinase 1), MECP2 (methyl CpG binding protein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCF1E (butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine A1 receptor), WRN (Werner syndrome, RecQ helicase-like), CXCR3 (chemokine (C-X-C motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen-activated protein kinase kinase kinase 5), CF1GA (chromogranin A (parathyroid secretory protein 1)), IAPP (islet amyloid polypeptide), RFIO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), PTF1LF1 (parathyroid hormone-like hormone), NRG1 (neuregulin 1), VEGFC (vascular endothelial growth factor C), ENPEP (glutamyl aminopeptidase (aminopeptidase A)), CEBPB (CCAAT/enhancer binding protein (C/EBP), beta), NAGLU (N-acetylglucosaminidase, alpha), F2RL3 (coagulation factor II (thrombin) receptor-like 3), CX3CL1 (chemokine (C-X3-C motif) ligand 1), BDKRB1 (bradykinin receptor B1), ADAMTS13 (ADAM metallopeptidase with thrombospondin type 1 motif, 13), ELANE (elastase, neutrophil expressed), ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2), CISF1 (cytokine inducible SF12-containing protein), GAST (gastrin), MYOC (myocilin, trabecular mesh work inducible glucocorticoid response), ATP1A2 (ATPase, Na+/K+ transporting, alpha 2 polypeptide), NF1 (neurofibromin 1), GJB1 (gap junction protein, beta 1, 32 kDa), MEF2A (myocyte enhancer factor 2A), VCL (vinculin), BMPR2 (bone morphogenetic protein receptor, type II (serine/threonine kinase)), TUBB (tubulin, beta), CDC42 (cell division cycle 42 (GTP binding protein, 25 kDa)), KRT18 (keratin 18), F1SF1 (heat shock transcription factor 1), MYB (v-myb myeloblastosis viral oncogene homolog (avian)), PRKAA2 (protein kinase, AMP-activated, alpha 2 catalytic subunit), ROCK2 (Rho-associated, coiled-coil containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor)), PRKG1 (protein kinase, cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CTF1 (cystathionase (cystathionine gamma-lyase)), CTSS (cathepsin S), VAV2 (vav 2 guanine nucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), IGFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase alpha 1), PPIA (peptidylprolyl isomerase A (cyclophilin A)), APOF1 (apolipoprotein FI (beta-2-glycoprotein I)), S100A8 (S100 calcium binding protein A8), IL11 (interleukin 11), ALOX15 (arachidonate 15-lipoxygenase), FBLN1 (fibulin 1), NR1F13 (nuclear receptor subfamily 1, group FI, member 3), SCD (stearoyl-CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory polypeptide), CF1 GB (chromogranin B (secretogranin 1)), PRKCB (protein kinase C, beta), SRD5A1 (steroid-5-alpha-reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1)), F1 SD11B2 (hydroxy steroid (11-beta) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4 (angiopoietin-like 4), KCNN4 (potassium intermediate/small conductance calcium-activated channel, subfamily N, member 4), PIK3C2A (phosphoinositide-3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (major histocompatibility complex, class II, DR beta 5), BNIP3 (BCL2/adeno virus E1B 19 kDa interacting protein 3), GCKR (glucokinase (hexokinase 4) regulator), S100A12 (S100 calcium binding protein A 12), PADI4 (peptidyl arginine deaminase, type IV), HSPA14 (heat shock 70 kDa protein 14), CXCR1 (chemokine (C-X-C motif) receptor 1), H19 (H19, imprinted maternally expressed transcript (non-protein coding)), KRTAP19-3 (keratin associated protein 19-3), insulin, RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)), RYR1 (ryanodine receptor 1 (skeletal)), CLOCK (clock homolog (mouse)), NGFR (nerve growth factor receptor (TNFR superfamily, member 16)), DBH (dopamine beta-hydroxylase (dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, alpha 4), CACNA1C (calcium channel, voltage-dependent, L type, alpha 1C subunit), PRKAG2 (protein kinase, AMP-activated, gamma 2 non-catalytic subunit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H, member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascular endothelial growth factor B), MEF2C (myocyte enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2), TNFRSF11 A (tumor necrosis factor receptor superfamily, member 11a, NFKB activator), HSPA9 (heat shock 70 kDa protein 9 (mortalin)), CYSLTR1 (cysteinyl leukotriene receptor 1), MAT1A (methionine adenosyltransferase I, alpha), OPRL1 (opiate receptor-like 1), IMPA1 (inositol(myo)-1(or 4)-monophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (prosome, macropain) subunit, alpha type, 6), PSMB8 (proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like 1 (cartilage glycoprotein-39)), ALDH1B1 (aldehyde dehydrogenase 1 family, member B1), PARP2 (poly (ADP-ribose) polymerase 2), STAR (steroidogenic acute regulatory protein), LBP (lipopolysaccharide binding protein), ABCC6 (ATP-binding cassette, sub-family C(CFTR/MRP), member 6), RGS2 (regulator of G-protein signaling 2, 24 kDa), EFNB2 (ephrin-B2), cystic fibrosis transmembrane conductance regulator (CFTR), GJB6 (gap junction protein, beta 6, 30 kDa), APOA2 (apolipoprotein A-II), AMPD1 (adenosine monophosphate deaminase 1), DYSF (dysferlin, limb girdle muscular dystrophy 2B (autosomal recessive)), FDFT1 (farnesyl-diphosphate farnesyltransferase 1), EDN2 (endothelin 2), CCR6 (chemokine (C-C motif) receptor 6), GJB3 (gap junction protein, beta 3, 31 kDa), IL1RL1 (interleukin 1 receptor-like 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (cadherin, EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophila)), F1IR (F11 receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaluronoglucosaminidase 1), ZNF259 (zinc finger protein 259), ATOX1 (ATX1 antioxidant protein 1 homolog (yeast)), ATF6 (activating transcription factor 6), KHK (ketohexokinase (fructokinase)), SAT1 (spermidine/spermine Nl-acetyltransferase 1), GGFI (gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl hydrolase)), TIMP4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solute carrier family 4, sodium bicarbonate cotransporter, member 4), PDE2A (phosphodiesterase 2 A, cGMP-stimulated), PDE3B (phosphodiesterase 3B, cGMP-inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin interacting protein), LIMS1 (LIM and senescent cell antigen-like domains 1), RFIOB (ras homolog gene family, member B), LY96 (lymphocyte antigen 96), FOXO1 (forkhead box 01), PNPLA2 (patatin-like phospholipase domain containing 2), TRH (thyrotropin-releasing hormone), GJC1 (gap junction protein, gamma 1, 45 kDa), SLC17A5 (solute carrier family 17 (anion/sugar transporter), member 5), FTO (fat mass and obesity associated), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC1 (proline/serine-rich coiled-coil 1), CASP12 (caspase 12 (gene/pseudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK (PX domain containing serine/threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles homolog 1 (Drosophila)), PBX4 (pre-B-cell leukemia homeobox 4), NUPR1 (nuclear protein, transcriptional regulator, 1), 15-Sep (15 kDa selenoprotein), CILP2 (cartilage intermediate layer protein 2), TERC (telomerase RNA component), GGT2 (gamma-glutamyltransferase 2), MT-COl (mitochondrially encoded cytochrome c oxidase I), UOX (urate oxidase, pseudogene), a CRISPR/Cas effector polypeptide, an enzymatically active CRISPR/Cas effector polypeptide (e.g., is capable of cleaving a target nucleic acid) and a CRISPR/Cas effector polypeptide that is not enzymatically active (e.g., does not cleave a target nucleic acid, but retains binding to the target nucleic acid). In some cases, the donor DNA encodes a wild-type version of any of the foregoing polypeptides; i.e., the donor DNA can encode a “normal” version that does not include a mutation(s) that results in reduced function, lack of function, or pathogenesis.

In some cases, the donor DNA comprises a nucleotide sequence encoding a fluorescent polypeptide. Suitable fluorescent proteins include, but are not limited to, green fluorescent protein (GFP) or variants thereof, blue fluorescent variant of GFP (BFP), cyan fluorescent variant of GFP (CFP), yellow fluorescent variant of GFP (YFP), enhanced GFP (EGFP), enhanced CFP (ECFP), enhanced YFP (EYFP), GFPS65T, Emerald, Topaz (TYFP), Venus, Citrine, mCitrine, GFPuv, destabilized EGFP (dEGFP), destabilized ECFP (dECFP), destabilised EYFP (dEYFP), mCFPm, Cerulean, T-Sapphire, CyPet, YPet, mKO, HcRed, t-HcRed, DsRed, DsRed2, DsRed-monomer, J-Red, dimer2, t-dimer2(12), mRFP1, pocilloporin, Renilla GFP, Monster GFP, paGFP, Kaede protein and kindling protein, Phycobiliproteins and Phycobiliprotein conjugates including B-Phycoerythrin, R-Phycoerythrin and Allophycocyanin. Other examples of fluorescent proteins include mHoneydew, mBanana, mOrange, dTomato, tdTomato, mTangerine, mStrawberry, mCherry, mGrape1, mRaspberry, mGrape2, m PI urn (Shaner et al. (2005) Nat. Methods 2:905-909), and the like. Any of a variety of fluorescent and colored proteins from Anthozoan species, as described in, e.g., Matz et al. (1999) Nature Biotechnol. 17:969-973, can be encoded.

In some cases, the donor DNA encodes an RNA, e.g., an siRNA, a microRNA, a short hairpin RNA (shRNA), an anti-sense RNA, a riboswitch, a ribozyme, an aptamer, a ribosomal RNA, a transfer RNA, and the like.

A donor DNA can include, in addition to a nucleotide sequence encoding one or more gene products (e.g., an RNA and/or a polypeptide), one or more transcriptional control elements, e.g., a promoter, an enhancer, and the like. In some cases, the transcriptional control element is inducible. In some cases, the promoter is reversible. In some cases, the transcriptional control element is constitutive. In some cases, the promoter is functional in a eukaryotic cell. In some cases, the promoter is a cell type-specific promoter. In some cases, the promoter is a tissue-specific promoter.

The nucleotide sequence of the donor DNA is typically not identical to the target nucleic acid (e.g., genomic sequence) that it replaces. Rather, the donor DNA may contain at least one or more single base changes, insertions, deletions, inversions or rearrangements with respect to the target nucleic acid (e.g., genomic sequence), so long as sufficient homology is present to support homology-directed repair (e.g., for gene correction, e.g., to convert a disease-causing base pair or a non-disease-causing base pair). In some cases, the donor DNA comprises a non-homologous sequence flanked by two regions of homology, such that homology-directed repair between the target DNA region and the two flanking sequences results in insertion of the non-homologous sequence at the target region. Donor DNA may also comprise a vector backbone containing sequences that are not homologous to the DNA region of interest (the target nucleic acid) and that are not intended for insertion into the DNA region of interest (the target nucleic acid). Generally, the homologous region(s) of a donor sequence will have at least 50% sequence identity to a target nucleic acid (e.g., a genomic sequence) with which recombination is desired. In certain cases, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 99.9% sequence identity is present. Any value between 1% and 100% sequence identity can be present, depending upon the length of the donor polynucleotide.

The donor DNA may comprise certain nucleotide sequence differences as compared to the target nucleic acid (e.g., genomic sequence), where such difference include, e.g. restriction sites, nucleotide polymorphisms, selectable markers (e.g., drug resistance genes, fluorescent proteins, enzymes etc.), etc., which may be used to assess for successful insertion of the donor DNA at the cleavage site or in some cases may be used for other purposes (e.g., to signify expression at the targeted genomic locus). In some cases, if located in a coding region, such nucleotide sequence differences will not change the amino acid sequence, or will make silent amino acid changes (i.e., changes which do not affect the structure or function of the protein). Alternatively, these sequences differences may include flanking recombination sequences such as FLPs, loxP sequences, or the like, that can be activated at a later time for removal of the marker sequence. In some cases, the donor DNA will include one or more nucleotide sequences to aid in localization of the donor to the nucleus of the recipient cell or to aid in the integration of the donor DNA into the target nucleic acid. For example, in some case, the donor DNA may comprise one or more nucleotide sequences encoding one or more nuclear localization signals (e.g. PKKKRKV (SEQ ID NO:1), VSRKRPRP (SEQ ID NO:2), QRKRKQ (SEQ ID NO:3), and the like (Frietas et al (2009) Cun-Genomics 10:550-7). In some cases, the donor DNA will include nucleotide sequences to recruit DNA repair enzymes to increase insertion efficiency. Fiuman enzymes involved in homology directed repair include MRN-CtIP, BLM-DNA2, Exo1, ERCC1, Rad51, Rad52, Ligase 1, RoIQ, PARP1, Ligase 3, BRCA2, RecQ/BLM-ToroIIIa, RTEL, Roid, and Roth (Verma and Greenburg (2016) Genes Dev. 30 (10): 1138-1154). In some cases, the donor DNA is delivered as reconstituted chromatin (Cruz-Becerra and Kadonaga (2020) eLife 2020; 9:e55780 DOI: 10.7554/eLife.55780).

In some cases, the ends of the donor DNA are protected (e.g., from exonucleolytic degradation) by any convenient method and such methods are known to those of skill in the art. For example, one or more dideoxynucleotide residues can be added to the 3′ terminus of a linear molecule and/or self complementary oligonucleotides can be ligated to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad Sci USA 84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues. As an alternative to protecting the termini of a linear donor DNA, additional lengths of sequence may be included outside of the regions of homology that can be degraded without impacting recombination.

HNS=Functional RNA Element

In certain embodiments, the donor/template comprises a coding sequence for a functional RNA element (ncRNAs, siRNA, shRNA, sgRNA, etc.).

In some embodiments, the heterologous nucleic acid sequences encode a functional non-translated RNA. In some embodiments, the functional non-translated RNA is an RNA aptamer or a ribozyme.

In some embodiments, the heterologous nucleic acid of the engineered retrons further includes a unique barcode to facilitate multiplexing. Barcodes may include one or more nucleotide sequences that are used to identify a nucleic acid or cell with which the barcode is associated. Such barcodes may be inserted for example, into the tip/loop region of the msd-encoded DNA. Barcodes can be 3-1000 or more nucleotides in length, 10-250 nucleotides in length, or 10-30 nucleotides in length, including any length within these ranges, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length.

In some embodiments, barcodes are also used to identify the position (viz., positional barcode) of a cell, colony, or sample from which a retron originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, the position of a tube in a rack, or the location of a sample in a laboratory. In particular, a barcode may be used to identify the position of a genetically modified cell containing a retron. The use of barcodes allows retrons from different cells to be pooled in a single reaction mixture for sequencing while still being able to trace a particular retron back to the colony from which it originated.

In addition, adapter sequences can be added to engineered retron to facilitate high-throughput amplification or sequencing. For example, a pair of adapter sequences can be added at the 5′ and 3′ ends of a retron construct to allow amplification or sequencing of multiple engineered retron simultaneously by the same set of primers.

HNS=Guide RNA

In some embodiments, the functional non-translated RNA is a CRISPR/Cas guide RNA (gRNA) specific for a target sequence in the mammalian cell. FIG. 1G depicts various configurations of a recombinant retron ncRNA which is modified by inserting a guide RNA at the 5′ end or the 3′ of the ncRNA. The guide RNA also may be provided in trans as a separate construct. In addition, guide RNAs may be placed at both ends of a recombinant retron ncRNA.

The skilled person will understand that selection of an appropriate guide RNA is informed by which RNA-guided nuclease is utilized.

A guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid. The term “guide RNA”, as used herein, refers to an RNA that comprises: i) an “activator” nucleotide sequence that binds to a CRISPR/Cas effector polypeptide (e.g., a class 2 CRISPR/Cas effector polypeptide such as a type II, type V, or type VI CRISPR/Cas endonuclease) and activates the CRISPR/Cas effector polypeptide; and ii) a “targeter” nucleotide sequence that comprises a nucleotide sequence that hybridizes with a target nucleic acid. The “activator” nucleotide sequence and the “targeter” nucleotide sequence can be on separate RNA molecules (e.g., a “dual-guide RNA”); or can be on the same RNA molecule (a “single-guide RNA”). A guide nucleic acid in some cases includes only ribonucleotides. In some cases, a guide nucleic acid includes both ribonucleotides and deoxyribonucleotides.

In some cases, a CRISPR/Cas guide RNA comprises one or more modifications, e.g., a base modification, a backbone modification, a sugar modification, etc., to provide the nucleic acid with a new or enhanced feature (e.g., improved stability, such as improved in vivo stability). Suitable nucleic acid modifications include, but are not limited to: 2′O-methyl modified nucleotides, 2′ fluoro modified nucleotides, locked nucleic acid (LNA) modified nucleotides, peptide nucleic acid (PNA) modified nucleotides, nucleotides with phosphorothioate linkages, and a 5′ cap (e.g., a 7-methyIguanyIate cap (m7G)). Suitable modified nucleic acid backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3-aIkyIene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Suitable oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be a basic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts (such as, for example, potassium or sodium), mixed salts and free acid forms are also included. A CRISPR-Cas guide RNA can also include one or more substituted sugar moieties. Suitable polynucleotides comprise a sugar substituent group selected from: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl. Particularly suitable are O((CH₂)_(n)O)_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON((CH₂)_(n)CH₃)₂, where n and m are from 1 to about 10. Other suitable polynucleotides comprise a sugar substituent group selected from: Ci to Cio lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A suitable modification includes 2′-methoxy ethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further suitable modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂.

Examples of various CRISPR/Cas effector proteins and CRISPR/Cas guide RNAs (as well as information regarding requirements related to protospacer adjacent motif (PAM) sequences present in targeted nucleic acids) can be found in the art, for example, see Jinek et al., Science. 2012 Aug. 17; 337(6096):816-21; Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Ma et al., Biomed Res Int. 2013; 2013:270805; Hou et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15644-9; Jinek et al., Elife. 2013; 2:e00471; Pattanayak et al., Nat Biotechnol. 2013 September; 31(9):839-43; Qi et al., Cell. 2013 Feb. 28; 152(5): 1173-83; Wang et al., Cell. 2013 May 9; 153(4):910-8; Auer et al., Genome Res. 2013 Oct. 31; Chen et al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e19; Cheng et al., Cell Res. 2013 October; 23(10):1163-71; Cho et al., Genetics. 2013 November; 195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 April; 41(7):4336-43; Dickinson et al., Nat Methods. 2013 October; 10(10):1028-34; Ebina et al., Sci Rep. 2013; 3:2510; Fujii et al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e187; Hu et al., Cell Res. 2013 November; 23(1):1322-5; Jiang et al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e188; Larson et al., Nat Protoc. 2013 November; 8(1):2180-96; Mali et al., Nat Methods. 2013 October; 10(10):957-63; Nakayama et al., Genesis. 2013 December; 51(12):835-43; Ran et al., Nat Protoc. 2013 November; 8(1):2281-308; Ran et al., Cell. 2013 Sep. 12; 154(6):1380-9; Upadhyay et al., G3 (Bethesda). 2013 Dec. 9; 3(12):2233-8; Walsh et al., Proc Natl Acad Sci USA. 2013 Sep. 24; 110(39):15514-5; Xie et al., Mol Plant. 2013 Oct. 9; Yang et al., Cell. 2013 Sep. 12; 154(6):1370-9; Briner et al., Mol Cell. 2014 Oct. 23; 56(2):333-9; and U.S. patents and patent applications: U.S. Pat. Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406; 8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006; 20140179770; 20140186843; 20140186919; 20140186958; 20140189896; 20140227787; 20140234972; 20140242664; 20140242699; 20140242700; 20140242702; 20140248702; 20140256046; 20140273037; 20140273226; 20140273230; 20140273231; 20140273232; 20140273233; 20140273234; 20140273235; 20140287938; 20140295556; 20140295557; 20140298547; 20140304853; 20140309487; 20140310828; 20140310830; 20140315985; 20140335063; 20140335620; 20140342456; 20140342457; 20140342458; 20140349400; 20140349405; 20140356867; 20140356956; 20140356958; 20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; all of which are hereby incorporated by reference in their entirety.

Examples and guidance related to type V or type VI CRISPR/Cas endonucleases and guide RNAs (as well as information regarding requirements related to protospacer adjacent motif (PAM) sequences present in targeted nucleic acids) can be found in the art, for example, see Zetsche et al., Cell. 2015 Oct. 22; 163(3):759-71; Makarova et al., Nat Rev Microbiol. 2015 November; 13(11):722-36; and Shmakov et al., Mol Cell. 2015 Nov. 5; 60(3):385-97.

C. Reverse Transcriptases (RTs)

Reverse transcriptases (RTs, also known as RNA-directed DNA polymerases) are enzymes present in all three domains of life, which are DNA polymerase using RNA as a template. Reverse transcriptases of the present disclosure are used to reverse transcribe template msd RNA into single-stranded msDNA.

The reverse transcriptase or a functional domain thereof that may be used in the instant invention includes prokaryotic and eukaryotic RT, provided that the RT functions within the host to generate a donor polynucleotide sequence from the RNA template (e.g., an RNA template from the retron transcript ncRNA).

In certain embodiments, suitable RT sequences (including amino acid sequences and the encoding polynucleotide sequences) are provided in Table A.

In certain embodiments, the nucleotide sequence of a native or wild-type RT is modified, for example, using known codon optimization techniques, so that expression within the desired host is optimized.

In certain embodiments, the RT domain of a reverse transcriptase is used in the present invention, so long as it is compatible with the engineered retron of the invention. The domain may include only the RNA-dependent DNA polymerase activity. In certain embodiments, the RT domain is non-mutagenic, i.e., does not cause mutation in the donor polynucleotide (e.g., during the reverse transcriptase process). In certain embodiments, the RT domain may be non-retron RT in origin, e.g., a viral RT or a human endogenous RTs. In certain embodiments, the RT domain is retron RT or DGRs RT. In certain embodiments, the RT may be less mutagenic than a counterpart wildtype RT. In certain embodiments, the RT is not mutagenic.

In some embodiments, a reverse transcriptase is encoded by a retron ret gene, which may accompany the cognate msr and msd loci and specifically recognize the secondary structure of the cognate ncRNA transcript.

In some embodiments, the RT may be obtained from prokaryotic or eukaryotic cells. Most reverse transcriptases (80%) can be phylogenetically clustered into three major lineages: group II introns, diversity-generating retroelements (DGRs), and retrons. Other clades of RTs include abortive infection (Abi) RTs, CRISPR-Cas-associated RTs, Group II-like (G2L), the unknown groups (UG), and rvt elements.

In some embodiments, the RT gene is a cognate RT, a retron RT from a species within the same species or clade of the cognate RT, or a retron RT not within the same clade of the cognate RT such as an unrelated RT or an engineered RT. In some embodiments, the non-retron related RT are RTs from group II introns, diversity-generating retroelements (DGRs), abortive infection (Abi) RTs, CRISPR-Cas-associated RTs, Group II-like (G2L), the unknown groups (UG), and rvt elements. See Mestre et al., Nucleic Acids Research, Volume 48, Issue 22, 16 Dec. 2020, Pages 12632-12647; and Mestere et al., UG/Abi: “A Highly Diverse Family of Prokaryotic Reverse Transcriptases Associated With Defense Functions,” doi.org/10.1101/2021.12.02.470933 (incorporated herein by reference).

In some embodiments, the RT are from clades related to retron/retron-like sequences. In some embodiments, the RT are selected from RTs provided in Table A. In some embodiments, the RT is not an RT associated with the sequences identified in Table X.

In prokaryotic retron systems, the RT gene is typically located downstream from the ncRNA (msr and msd) locus. In the engineered retron, the RT position can differ from the natural or wild type retrons. In some embodiments, the RT gene can be provided in cis, such as either upstream or downstream of the msr locus or the msd locus. In certain embodiments, the RT gene is provided in trans, such as provided separately in a vector of the vector system described herein, wherein the ncRNA coding msr and msd sequences are provided in a different vector of the vector system described herein.

In some embodiments, the RT is modified (e.g., insertion, deletion, and/or substitution of one or more nucleotide(s)) or codon optimized to enhance activity or processivity.

In certain embodiments, a cryptic stop signal is removed from the RT thereby allowing generation of longer ssDNAs.

In certain embodiments, the RT is from a retron which encodes msDNA as described in U.S. Pat. Nos. 6,017,737; 5,849,563; 5,780,269; 5,436,141; 5,405,775; 5,320,958; CA2,075,515; all of which are incorporated by reference herein in their entireties.

In some embodiments, the engineered retron further comprises a polynucleotide (e.g., a DNA molecule) encoding a reverse transcriptase (RT) or a portion thereof. In some embodiments, the encoded RT or portion thereof is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA.

In some embodiments, the polynucleotide (e.g., a DNA molecule) encoding the RT comprises a polynucleotide listed in Table A, or a polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polynucleotide listed in Table A.

In some embodiments, the polynucleotide encoding the RT encodes a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or a polypeptide of Table C.

In some embodiments, the polynucleotide encoding the RT does not comprise a polynucleotide listed in Table X.

Once translated, the RT binds the ncRNA template downstream from the msd locus, forming an RT-RNA complex, and initiating reverse transcription of the RNA towards its 5′ end. Accordingly, in certain aspects the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a. a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b. a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c. a reverse transcriptase (RT), or a domain thereof comprising: i) a polypeptide listed in Table A, or a polypeptide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to a polypeptide listed in Table A; and/or ii) a polypeptide listed in Table C. In some embodiments, the RT does not comprise a polypeptide listed in Table X.

In certain aspects, the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA, b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus; upstream of the msr locus; upstream of the msd locus; and downstream of the msd locus; and c) a reverse transcriptase (RT), or a portion thereof, wherein the RT is capable of synthesizing a DNA copy of at least a portion of the msd locus encoding the msDNA, and wherein the ncRNA and/or the RT is any one of the invention described herein.

In certain aspects, the disclosure relates to an engineered nucleic acid-enzyme construct comprising: a) a non-coding RNA (ncRNA) comprising: i) an msr locus encoding the msr RNA portion of a multi-copy single-stranded DNA (msDNA); and ii) an msd locus encoding the msd RNA portion of the msDNA; b) a heterologous nucleic acid inserted at or within a location selected from: the msd locus, upstream of the msr locus, upstream of the msd locus, and downstream of the msd locus; and c) a reverse transcriptase (RT) or a domain thereof: wherein the RT comprises: i) an RT listed in Table A, or an RT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an RT listed in Table A; and/or ii) a consensus sequence listed in Table C; and wherein, the RT does not comprise a sequence from Table X.

In some embodiments of the nucleic-acid enzyme constructs described herein, the ncRNA comprises: i) an ncRNA listed in Table B, or an ncRNA having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to an ncRNA listed in Table B; and/or optionally wherein the ncRNA is not an ncRNA from the retons of Table X.

In some embodiments, the RT is linked to components such as RNA-guided and non-RNA guided nucleases. The linked maybe via s peptide bond or a short linker peptide in a fusion protein. Suitable linker peptides include flexible linkers such as those comprising G or S repeats, such as G₄S repeat units or GS repeat units, with 1-20 repeats, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 repeats.

In certain embodiments, the RT is chemically linked or conjugated to the RNA-guided and non-RNA guided nucleases via non-peptide bonds. Such protein conjugates may be delivered directly to a host cell, either together with the nucleic acid component of the engineered retron described herein, or separately.

In some embodiments, the RT is linked to a DNA-repair modulating biomolecule (e.g., NHEJ peptide inhibitors.

D. Programmable Nucleases (RNA-Guided Nucleases)

In certain embodiments, the engineered retron (e.g., an engineered nucleic acid construct or engineered nucleic acid-enzyme construct as described herein) may comprise or encode, as one heterologous nucleic acid, a guide RNA (gRNA) suitable for guiding a nuclease to target a particular genomic sequence to be modified. The gRNA includes sequences complementary to a genomic sequence, and therefore can mediate binding of the nuclease-gRNA complex to the genomic target site by hybridization between the guide sequence and the target site sequence.

In certain embodiments, the gRNA can be linked to the ncRNA and/or the msDNA encoded by the engineered retron described herein at the 5′ end of the ncRNA and/or the msDNA. In certain embodiments, the gRNA can be linked to the ncRNA and/or the msDNA encoded by the engineered retron described herein at the 3′ end of the RNA of the ncRNA and/or the msDNA after reverse transcription.

In some embodiments, the nuclease that can form a complex with the gRNA can be any one of the art-recognized clustered regularly interspersed short palindromic repeats (CRISPR) system Cas effector enzymes, which are useful for, e.g., genome editing, including genome editing in mammalian cells or human cells.

For example, a gRNA that can be loaded into a Cas9 or variant thereof may be encoded by the engineered retron, such that the gRNA is transcribed as part of the msDNA. In some embodiments, the gRNA may be linked to the 5′ end of the a1 region of the msr-encoded sequence in the retron ncRNA, as well as the msDNA after reverse transcription. In some embodiments, the gRNA may be part of the modified msr-region present in the ncRNA and the msDNA produced after reverse transcription (viz., the encoded gRNA is not degraded by the synthesis of the msDNA through reverse transcription of the ncRNA).

Any art recognized CRISPR/Cas effector enzymes or variants thereof (“Cas enzymes”) known to be useful for CRISPR-based genome editing can be used with the engineered retron, though such Cas enzymes may not necessarily be part of the engineered retron, and can (but is not required to) be provided separately. For example, the Cas enzymes can be provided as part of the vector system described herein. The coding sequence of the suitable Cas enzyme can be present on the same vector that provides the engineered retron, or on different vectors. When the engineered retron and the Cas enzymes are present on the same vector, they may be under the transcriptional control of the same promoter, enhancer, or other transcriptional regulatory elements, or may be separately regulated by different promoters, enhancers, and/or other transcriptional regulatory sequences in the vector system.

In some embodiments, the Cas enzyme is a Class 2, Type II CRISPR/Cas effector enzyme, such as Cas9. In some embodiments, the Cas9 is from Streptococcus pyogenes (SpCas9), and the gRNA encoded by the engineered retron comprises both the crisprRNA (crRNA) and the tracrRNA linked together as the single guide RNA (gRNA).

In some embodiments, the Cas9 is from Staphylococcus aureus Cas9 (SaCas9), or an engineered variant thereof such as SaCas9-HF (a high-fidelity variant with genome-wide activity), KKHSaCas9 (which recognizes a 5′-NNGRRT-3′ PAM, and has a 2-4× broader range of target sites in the human genome than the wildtype SaCas9), and microABE1744 (an engineered SaCas9 variant adapted for use in adenine base editing (ABE), with significantly improved on-target editing compared to other nucleases, with a reduced RNA off-target footprint).

In some embodiments, the Cas9 is from Streptococcus thermophilus (StCas9), Neisseria meningitidis (NmCas9), Francisella novicida (FnCas9), or Campylobacter jejuni (CjCas9).

In some embodiments, the Cas9 is from Streptococcus canis (ScCas9), with a less stringent PAM sequence requirement of 5′-NNG-3′ (instead of the more stringent 5′-NGG-3′ for SpCas9).

In some embodiments, the Cas9 is from Staphylococcus auricularis (SauriCas9) (which recognizes a 5′-NNGG-3′ PAM sequence, has high editing activity).

In some embodiments, the Cas enzyme is a Cas9 variant with mutated catalytic domain that retains cleavage specificity, but only nicks a single DNA strand at a desired target sequence instead of creating a double-strand break (DSB). Two such Cas9 nickase variants targeting different strands of the same target sequence may be used together to increase fidelity of creating the DSB, each using a different gRNA that can be provided by the engineered retron.

In some embodiments, the Cas enzyme is a high fidelity Cas9 variant with weakened DNA phosphate backbone interaction (such as SpCas9-HF1) that displays genome-wide specificity and undetectable off-target effects.

In some embodiments, the Cas enzyme is a Cas9 variant known as eSpCas9, which has weakened interactions between the eSpCas9 and its gRNAs with non-exact complementarity with the target DNA sequence, thus providing improved specificity and lower off-target editing rates.

In some embodiments, the Cas enzyme is a hyper-accurate Cas9 variant (HypaCas9), which improves proofreading before cleavage, and thus drastically reduces off-target cleavage.

In some embodiments, the Cas enzyme is a Cas9 variant (FokI-Fused dCas9), which combines the DNA recognition ability by dCas9 with the specificity of an active nuclease FokI. The resulting nuclease cuts the target sequence only after dimerization, which is more difficult to occur at off-target sites, thus resulting in enhanced specificity.

In some embodiments, the Cas enzyme is a Cas9 variant xCas9, which recognizes a broad range of PAM sequences, thus increasing the target sites to 1 in 4 in the genome. In addition, the xCas9 variant also exhibits lower off-target rates than the commonly used SpCas9.

In some embodiments, the Cas enzyme is a Cas9 variant with altered PAM sequence specificities, including the SpG variant with an expanded target range of PAM sequences, and the SpRY variant that can target almost all PAM sequences.

In some embodiments, the Cas enzyme is dCas9 having inactivated catalytic nuclease domains while maintaining the recognition domains that allow guide RNA-mediated targeting to specific DNA sequences. The dCas9 may be further linked to a functional domain with a distinct biological function, such as transcriptional activation/depression, DNA methylation, demethylation, endonuclease (such as FokI), or fluorescent dye. Representative (non-limiting) dCas9-linked functional domains include including dCas9-SAM, dCas9-SunTag, dCas9-VPR, and dCas9-KREB.

In some embodiments, the dCas9 is fused with a catalytic enzyme with cytidine deaminase activity, which converts GC basepair to AT basepair.

In some embodiments, the dCas9 is fused with an engineered RNA adenosine deaminase, which converts AT basepair to GC basepair.

In some embodiments, the Cas enzyme is a Class 2, Type V CRISPR/Cas effector enzyme, such as Cas12a (Cpf1) (Type V-A), Cas12b (C2c1) (Type V-B), Cas12c (C2c3) (Type V-C), Cas12d (CasY) (Type V-D), Cas12e (CasX) (Type V-E), Cas12f (Cas14, C2c10) (Type V-F), Cas12g (Type V-G), Cas12h (Type V-H), Cas12i (Type V-I), Cas12k (C2c5) (Type V-K), or C2c4/C2c8/C2c9 (Type V-U).

In some embodiments, the Cas enzyme is Cas12a or Cpf1 (CRISPR from Prevotella and Francisella 1). Unlike Cas9, Cas12a is well-suited for targeting AT-rich DNA sequences because of its AT-rich PAM sequences. In some embodiments, the Cas12a is FnCas12a (that recognizes PAM sequence 5′-TTN-3′), or AsCas12a or LbCas12a (that recognize 5′-TTTV-3′ PAM sequence), where V is A, G, or C nucleotide. Further, Cas12a creates staggered double-stranded breaks in the target DNA, rather than the blunt-ends generated by SpCas9, thus rendering it more useful for HDR repair. In this embodiment, the subject engineered retron encodes a gRNA suitable for use for Cas12a, in that the gRNA does not require a tracer RNA, and only requires the crRNA.

In some embodiments, the Cas enzyme is a Cas12a variant from Acidaminococcus sp. (enAsCas12a), which has an expanded target range of PAM sequences and significantly higher editing activity compared to wild type Cas12a.

In some embodiments, the Cas enzyme is a high-fidelity Cas12a variant (enAsCas12a-HF1) that reduces off-target editing.

In some embodiments, the Cas enzyme is Cas12b or C2c1. In some embodiments, the Cas12b is form Alicyclobacillus acidoterrestris (AacCas12b), or from Alicyclobacillus acidiphilus Cas12b (AapCas12b).

In some embodiments, the Cas enzyme is a Cas12b variant that functions at 37° C., such as one form Bacillus hisashii (BhCas12b).

In some embodiments, the Cas enzyme is a BhCas12b variant with higher specificity than SpCas9.

In some embodiments, the Cas enzyme is CasX or Cas12d.

In some embodiments, the Cas enzyme is CasY or Cas12e, and the subject engineered retron encodes a short-complementarity untranslated RNA (scoutRNA) together with crRNA (rather than tracrRNA used in other CRISPR-Cas systems).

In some embodiments, the Cas enzyme is Cas14, from archaea bacteria. Cas14 targets single-stranded (ss) DNA target sequences, does not require PAM sequence for activation, and has collateral activity (i.e., cuts other non-target ssDNA strands non-specifically upon binding the target sequence). Unlike Cas12a, Cas14a requires high fidelity complementarity to the target ssDNA, and is very sensitive to internal seed region mismatches of the ssDNA target substrate.

In some embodiments, the gRNA nuclease is an engineered RNA-guided Fok1 nuclease. RNA-guided Fok1 nucleases comprise fusions of inactive Cas9 (dCas9) and the Fok1 endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fok1. For a description of engineered RNA-guided Fold nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

In other embodiments, the RNA-guided nuclease can be a non-CRISPER/Cas related nuclease such as transposon-encoded nucleases, IscBs, IscR, or TnpBs.

In some embodiments, the Cas enzyme is a Cas9

In some embodiments, the retron-based editing systems described herein can include any Cas9 equivalent. A Cas9 equivalent is a Cas9-like protein that provides the same or substantially the same function as Cas9. For instance, if Cas9 refers to a type II enzyme of the CRISPR-Cas system, a Cas9 equivalent can refer to a type V or type VI enzyme of the CRISPR-Cas system.

For example, Cas12e (CasX) is a Cas9 equivalent that reportedly has the same function as Cas9 but which evolved through convergent evolution. Thus, the Cas12e (CasX) protein is contemplated to be used with the retron-based editor systems described herein. In addition, any variant or modification of Cas12e (CasX) is conceivable and within the scope of the present disclosure.

In some embodiments, Cas9 equivalents may refer to Cas12e (CasX) or Cas12d (CasY), which have been described in, for example, Burstein et al., “New CRISPR-Cas systems from uncultivated microbes.” Cell Res. 2017 Feb. 21. doi: 10.1038/cr.2017.21, the entire contents of which is hereby incorporated by reference. Using genome-resolved metagenomics, a number of CRISPR-Cas systems were identified, including the first reported Cas9 in the archaeal domain of life. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems were discovered, CRISPR-Cas12e and CRISPR-Cas12d, which are among the most compact systems yet discovered. In some embodiments, Cas9 refers to Cas12e, or a variant of Cas12e. In some embodiments, Cas9 refers to a Cas12d, or a variant of Cas12d. It should be appreciated that other RNA-guided DNA binding proteins may be used as a nucleic acid programmable DNA binding protein (napDNAbp), and are within the scope of this disclosure.

In some embodiments, the Cas9 equivalent comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the napDNAbp is a naturally-occurring Cas12e (CasX) or Cas12d (CasY) protein. In some embodiments, the napDNAbp comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a wild-type Cas moiety or any Cas moiety provided herein.

In various embodiments, the nucleic acid programmable DNA binding proteins include, without limitation, Cas9 (e.g., dCas9 and nCas9), Cas12e (CasX), Cas12d (CasY), Cas12a (Cpf1), Cas12b1 (C2c1), Cas13a (C2c2), Cas12c (C2c3), Argonaute, and Cas12b1. One example of a nucleic acid programmable DNA-binding protein that has different PAM specificity than Cas9 is Clustered Regularly Interspaced Short Palindromic Repeats from Prevotella and Francisella 1 (i.e, Cas12a (Cpf1)). Similar to Cas9, Cas12a (Cpf1) is also a Class 2 CRISPR effector, but it is a member of type V subgroup of enzymes, rather than the type II subgroup. It has been shown that Cas12a (Cpf1) mediates robust DNA interference with features distinct from Cas9. Cas12a (Cpf1) is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif (TTN, TTTN, or YTN). Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, two enzymes from Acidaminococcus and Lachnospiraceae are shown to have efficient genome-editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example Yamano et al., “Crystal structure of Cpf1 in complex with guide RNA and target DNA.” Cell (165) 2016, p. 949-962; the entire contents of which is hereby incorporated by reference.

In still other embodiments, the Cas protein may include any CRISPR associated protein, including but not limited to, Cas12a, Cas12b1, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified versions thereof.

In various other embodiments, the RNA-guided nuclease can be any of the following proteins: a Cas9, a Cas12a (Cpf1), a Cas12e (CasX), a Cas12d (CasY), a Cas12b1 (C2c1), a Cas13a (C2c2), a Cas12c (C2c3), a GeoCas9, a CjCas9, a Cas12g, a Cas12h, a Cas12i, a Cas13b, a Cas13c, a Cas13d, a Cas14, a Csn2, an xCas9, an SpCas9-NG, or an Argonaute (Ago) domain, or a variant thereof.

Amino acid sequences of RNA-guided nucleases are readily available and known in the art. Exemplary RNA-guided nucleases and their amino acid sequences can be found, for example, in WO 2017/070633, US 2020/0010835, US 2022/0204975, U.S. Ser. No. 11/071,790, WO 2020/191233, U.S. Ser. No. 11/447,770, U.S. Ser. No. 10/858,639, and U.S. Ser. No. 10/947,530, each of which are incorporated herein by reference in their entireties.

E. Programmable Nucleases (Other)

In addition to the CRISPR/Cas system, the subject engineered retron can also be used in combination with sequence-specific nucleases that do not use a guide RNA to recognize a target sequence, such as non-CRISPR/Cas sequence-specific nucleases including TALENs, ZFNs, meganucleases, and restriction enzymes, as well as other sequence-specific nucleases that use other RNA guides, such as transposon-encoded IscBs, IscR, or TnpBs.

For example, the subject engineered retron may encode or provide a msDNA that can serve as a donor or template sequence for HDR-mediated genome editing. Optionally, the RT of the engineered retron is fused to such sequence-specific nuclease, such that the msDNA, by way of being generated by the RT close to the site of HDR-mediated genome editing, can be more efficiently participate in the HDR-mediated genome editing.

In some embodiments, the non-CRISPR/Cas sequence-specific nuclease is or comprises a TALE Nuclease, a TALE nickase, Zinc Finger (ZF) Nuclease, ZF Nickase, meganuclease, or a combination thereof. In some embodiments, the non-CRISPR/Cas sequence-specific nuclease is or includes two, three, four, or more of an independently selected TALE Nuclease, TALE nickase, Zinc Finger (ZF) Nuclease, ZF Nickase, Meganuclease, restriction enzymes or a combination thereof. In some embodiments, the combination is or comprises a TALE Nuclease/a ZF Nuclease; a TALE Nickase/a ZF nickase.

In some embodiments, the non-CRISPR/Cas sequence-specific nuclease is or comprises a TALE Nuclease (Transcription Activator-Like Effector Nucleases (TALEN)). TALENs are restriction enzymes engineered to cut specific target DNA sequences. TALENs comprise a TAL effector (TALE) DNA-binding domain (which binds at or close to the target DNA), fused to a DNA cleavage domain which cuts target DNA. TALEs are engineered to bind to practically any desired DNA sequence. Thus in some embodiments, the TALEN comprises an N-terminal capping region, a DNA binding domain which may comprise at least one or more TALE monomers or half-monomers specifically ordered to target the genomic locus of interest, and a C-terminal capping region, wherein these three parts are arranged in a predetermined N-terminus to C-terminus orientation. Optionally, the TALEN includes at least one or more regulatory or functional protein domains.

In some embodiments, the TALE monomers or half monomers may be variant TALE monomers derived from natural or wild type TALE monomers but with altered amino acids at positions usually highly conserved in nature, and in particular have a combination of amino acids as RVDs that do not occur in nature, and which may recognize a nucleotide with a higher activity, specificity, and/or affinity than a naturally occurring RVD. The variants may include deletions, insertions and substitutions at the amino acid level, and transversions, transitions and inversions at the nucleic acid level at one or more locations. The variants may also include truncations.

In some embodiments, the TALE monomer/half monomer variants include homologous and functional derivatives of the parent molecules. In some embodiments, the variants are encoded by polynucleotides capable of hybridizing under high stringency conditions to the parent molecule-encoding wild-type nucleotide sequences.

In some embodiments, the DNA binding domain of the TALE has at least 5 of more TALE monomers and at least one or more half-monomers specifically ordered or arranged to target a genomic locus of interest. The construction and generation of TALEs or polypeptides of the invention may involve any of the methods known in the art.

Naturally occurring TALEs or “wild type TALEs” are nucleic acid binding proteins secreted by numerous species of proteobacteria. TALEs contain a nucleic acid binding domain composed of tandem repeats of highly conserved monomer polypeptides that are predominantly 33, 34 or 35 amino acids in length and that differ from each other mainly in amino acid positions 12 and 13. A general representation of a TALE monomer which is comprised within the DNA binding domain is X1-11-(X12X13)-X14-33 or 34 or 35, where the subscript indicates the amino acid position and X represents any amino acid. X12X13 indicate the RVDs. In some polypeptide monomers, the variable amino acid at position 13 is missing or absent and in such monomers, the RVD consists of a single amino acid. In such cases the RVD may be alternatively represented as X*, where X represents X12 and (*) indicates that X13 is absent. The DNA binding domain may comprise several repeats of TALE monomers and this may be represented as (X1-11-(X12X13)-X14-33 or 34 or 35)z, where z is optionally at least 5-40, such as 10-26.

The TALE monomers have a nucleotide binding affinity that is determined by the identity of the amino acids in its RVD. Polypeptide monomers with an RVD of NI preferentially bind to adenine (A), monomers with an RVD of NG preferentially bind to thymine (T), monomers with an RVD of HD preferentially bind to cytosine (C), monomers with an RVD of NN preferentially bind to both adenine (A) and guanine (G), monomers with an RVD of IG preferentially bind to T, monomers with an RVD of NS recognize all four base pairs and may bind to A, T, G or C. Thus, the number and order of the polypeptide monomer repeats in the nucleic acid binding domain of a TALE determines its nucleic acid target specificity. The structure and function of TALEs is further described in, for example, Moscou et al., Science 326:1501 (2009); Boch et al., Science 326:1509-1512 (2009); and Zhang et al., Nature Biotechnology 29:149-153 (2011), each of which is incorporated by reference in its entirety.

In some embodiments, the TALE is a dTALE (or designerTALE), see Zhang et al., Nature Biotechnology 29:149-153 (2011), incorporated herein by reference.

In some embodiments, the TALE monomer comprises an RVD of HN or NH that preferentially binds to guanine, and the TALEs have high binding specificity for guanine containing target nucleic acid sequences. In come embodiments, polypeptide monomers having RVDs RN, NN, NK, SN, NH, KN, HN, NQ, HH, RG, KH, RH and SS preferentially bind to guanine. In some embodiments, polypeptide monomers having RVDs RN, NK, NQ, HH, KH, RH, SS and SN preferentially bind to guanine. In some embodiments, polypeptide monomers having RVDs HH, KH, NH, NK, NQ, RH, RN and SS preferentially bind to guanine. In some embodiments, the RVDs that have high binding specificity for guanine are RN, NH RH and KH. In some embodiments, polypeptide monomers having an RVD of NV preferentially bind to adenine and guanine as do monomers having the RVD HN. Monomers having an RVD of NC preferentially bind to adenine, guanine and cytosine, and monomers having an RVD of S (or S*), bind to adenine, guanine, cytosine and thymine with comparable affinity. In more embodiments, monomers having RVDs of H*, HA, KA, N*, NA, NC, NS, RA, and S* bind to adenine, guanine, cytosine and thymine with comparable affinity. Such polypeptide monomers allow for the generation of degenerative TALEs able to bind to a repertoire of related, but not identical, target nucleic acid sequences.

In certain embodiments, the TALE polypeptide has a nucleic acid binding domain containing polypeptide monomers arranged in a predetermined N-terminus to C-terminus order such that each polypeptide monomer binds to a nucleotide of a predetermined target nucleic acid sequence, and where at least one of the polypeptide monomers has an RVD of HN or NH and preferentially binds to guanine, an RVD of NV and preferentially binds to adenine and guanine, an RVD of NC and preferentially binds to adenine, guanine and cytosine or an RVD of S and binds to adenine, guanine, cytosine and thymine.

In some embodiments, each polypeptide monomer of the nucleic acid binding domain that binds to adenine has an RVD of NI, NN, NV, NC or S.

In certain embodiments, each polypeptide monomer of the nucleic acid binding domain that binds to guanine has an RVD of HN, NH, NN, NV, NC or S.

In certain embodiments, each polypeptide monomer of the nucleic acid binding domain that binds to cytosine has an RVD of HD, NC or S.

In some embodiments, each polypeptide monomer that binds to thymine has an RVD of NG or S.

In some embodiments, each polypeptide monomer of the nucleic acid binding domain that binds to adenine has an RVD of NI.

In certain embodiments, each polypeptide monomer of the nucleic acid binding domain that binds to guanine has an RVD of HN or NH.

In certain embodiments, each polypeptide monomer of the nucleic acid binding domain that binds to cytosine has an RVD of HD.

In some embodiments, each polypeptide monomer that binds to thymine has an RVD of NG.

In certain embodiments, the RVDs that have a specificity for adenine are NI, RI, KI, HI, and SI.

In certain embodiments, the RVDs that have a specificity for adenine are HN, SI and RI, most preferably the RVD for adenine specificity is SI.

In certain embodiments, the RVDs that have a specificity for thymine are NG, HG, RG and KG.

In certain embodiments, the RVDs that have a specificity for thymine are KG, HG and RG, most preferably the RVD for thymine specificity is KG or RG.

In certain embodiments, the RVDs that have a specificity for cytosine are HD, ND, KD, RD, HH, YG and SD.

In certain embodiments, the RVDs that have a specificity for cytosine are SD and RD.

FIG. 4B of WO 2012/067428 provides representative RVDs and the nucleotides they target, the entire content of which is hereby incorporated herein by reference.

In certain embodiments, the variant TALE monomers may comprise any of the RVDs that exhibit specificity for a nucleotide as depicted in FIG. 4A of WO2012/067428. All such TALE monomers allow for the generation of degenerative TALEs able to bind to a repertoire of related, but not identical, target nucleic acid sequences.

In certain embodiments, the RVD SH may have a specificity for G, the RVD IS may have a specificity for A, and the RVD IG may have a specificity for T.

In certain embodiments, the RVD NT may bind to G and A. In certain embodiments, the RVD NP may bind to A, T and C. In certain embodiments, at least one selected RVD may be NI, HD, NG, NN, KN, RN, NH, NQ, SS, SN, NK, KH, RH, HH, KI, HI, RI, SI, KG, HG, RG, SD, ND, KD, RD, YG, HN, NV, NS, HA, S*, N*, KA, H*, RA, NA or NC.

The predetermined N-terminal to C-terminal order of the one or more polypeptide monomers of the nucleic acid or DNA binding domain determines the corresponding predetermined target nucleic acid sequence to which the TALE or polypeptides of the invention may bind.

As used herein the monomers and at least one or more half monomers are “specifically ordered to target” the genomic locus or gene of interest. In plant genomes, the natural TALE-binding sites always begin with a thymine (T), which may be specified by a cryptic signal within the non-repetitive N-terminus of the TALE polypeptide; in some cases this region may be referred to as repeat 0. In animal genomes, TALE binding sites do not necessarily have to begin with a thymine (T) and polypeptides of the invention may target DNA sequences that begin with T, A, G or C. The tandem repeat of TALE monomers always ends with a half-length repeat or a stretch of sequence that may share identity with only the first 20 amino acids of a repetitive full length TALE monomer and this half repeat may be referred to as a half-monomer (FIG. 8 of WO 2012/067428). Therefore, it follows that the length of the nucleic acid or DNA being targeted is equal to the number of full monomers plus two (see FIG. 44 of WO 2012/067428).

In certain embodiments, nucleic acid binding domains are engineered to contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more polypeptide monomers arranged in a N-terminal to C-terminal direction to bind to a predetermined 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotide length nucleic acid sequence.

In certain embodiments, nucleic acid binding domains are engineered to contain 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or more full length polypeptide monomers that are specifically ordered or arranged to target nucleic acid sequences of length 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27 and 28 nucleotides, respectively. In certain embodiments, the polypeptide monomers are contiguous. In some embodiments, half-monomers may be used in the place of one or more monomers, particularly if they are present at the C-terminus of the TALE.

Polypeptide monomers are generally 33, 34 or 35 amino acids in length. With the exception of the RVD, the amino acid sequences of polypeptide monomers are highly conserved or as described herein, the amino acids in a polypeptide monomer, with the exception of the RVD, exhibit patterns that effect TALE activity, the identification of which may be used in preferred embodiments of the invention.

In certain embodiments, when the DNA binding domain may comprise (X1-11-X12X13-X14-33 or 34 or 35)z, wherein X1-11 is a chain of 11 contiguous amino acids, wherein X12X13 is a repeat variable diresidue (RVD), wherein X14-33 or 34 or 35 is a chain of 21, 22 or 23 contiguous amino acids, wherein z is at least 5 to 26, then the preferred combinations of amino acids are LTLD or LTLA or LTQV at X-4, or EQHG or RDHG at positions X30-33 or X31-34 or X32-35. Furthermore, other amino acid combinations of interest in the monomers are LTPD at X1-4 and NQALE at XI 6-20 and DHG at X32-34 when the monomer is 34 amino acids in length. When the monomer is 33 or 35 amino acids long, then the corresponding shift occurs in the positions of the contiguous amino acids NQALE and DHG. In certain embodiments, NQALE is at X15-19 or X17-21 and DHG is at X31-33 or X33-35.

In certain embodiments, amino acid combinations of interest in the monomers, are LTPD at X1-4 and KRALE at X16-20 and AHG at X32-34 or LTPE at X1-4 and KRALE at XI 6-20 and DHG at X32-34 when the monomer is 34 amino acids in length. When the monomer is 33 or 35 amino acids long, the corresponding shift occurs in the positions of the contiguous amino acids KRALE, AHG and DHG. In certain embodiments, the positions of the contiguous amino acids may be (LTPD at X1-4 and KRALE at X15-19 and AHG at X31-33) or (LTPE at X1-4 and KRALE at X15-19 and DHG at X31-33) or (LTPD at X1-4 and KRALE at X17-21 and AHG at X33-35) or (LTPE at X1-4 and KRALE at X17-21 and DHG at X33-35).

In certain embodiments, contiguous amino acids [NGKQALE] are present at positions X14-20 or X13-19 or X15-21. These representative positions put forward various embodiments of the invention and provide guidance to identify additional amino acids of interest or combinations of amino acids of interest in all the TALE monomers (see FIGS. 24A-24F, and 25 of WO 2012/067428).

Exemplary amino acid sequences of conserved portions of polypeptide monomers are provided below. The position of the RVD in each sequence is represented by XX or by X* (wherein (*) indicates that the RVD is a single amino acid and residue 13 (X13) is absent).

LTPAQVVAIASXXGGKQALETVQRLLPVLCQDHG LTPAQVVAIASX*GGKQALETVQRLLPVLCQDHG LTPDQVVAIANXXGGKQALATVQRLLPVLCQDHG LTPDQVVAIANXXGGKQALETLQRLLPVLCQDHG LTPDQVVAIANXXGGKQALETVQRLLPVLCQDHG LTPDQVVAIASXXGGKQALATVQRLLPVLCQDHG LTPDQVVAIASXXGGKQALETVQRLLPVLCQDHG LTPDQVVAIASXXGGKOALETVORVLPVLCODHG LTPEQVVAIASXXGGKQALETVQRLLPVLCQAHG LTPYQVVAIASXXGSKQALETVQRLLPVLCQDHG LTREQVVAIASXXGGKQALETVQRLLPVLCQDHG LSTAQVVAIASXXGGKQALEGIGEQLLKLRTAPYG LSTAQVVAVASXXGGKPALEAVRAQLLALRAAPYG

A further listing of TALE monomers excluding the RVDs which may be denoted in a sequence (X1-11-X14-34 or X1-11-X14-35), wherein X is any amino acid and the subscript is the amino acid position is provided in FIG. 24A-F of WO 2012/067428, which is incorporated herein by reference.

In certain embodiments, TALE polypeptide binding efficiency is increased by including amino acid sequences from the “capping regions” that are directly N-terminal or C-terminal of the DNA binding region of naturally occurring TALEs into the engineered TALEs at positions N-terminal or C-terminal of the engineered TALE DNA binding region. Thus, in certain embodiments, the TALE polypeptides described herein further comprise an N-terminal capping region and/or a C-terminal capping region.

An exemplary amino acid sequence of a N-terminal capping region is:

MDPIRSRTPSPARELLSGPQPDGVQPTADRGVSPPAGGPL DGLPARRTMSRTRLPSPPAPSPAFSADSFSDLLRQFDPSL FNTSLFDSLPPFGAHHTEAATGEWDEVQSGLRAADAPPPT MRVAVTAARPPRAKPAPRRRAAQPSDASPAAQVDLRTLGY SQQQQEKIKPKVRSTVAQHHEALVGHGFTHAHIVALSQHP AALGTVAVKYQDMIAALPEATHEAIVGVGKQWSGARALEA LLTVAGELRGPPLQLDTGQLLKIAKRGGVTAVEAVHAWRN ALTGAPLN

An exemplary amino acid sequence of a C-terminal capping region is:

REALESIVAQLSRPDPALAALTNDHLVALACLGGRPALDA VKKGLPHAPALIKRTNRRIPERTSHRVADHAQWRVLGFFQ CHSHPAQAFDDAMTQFGMSRHGLLQLFRRVGVTELEARSG TLPPASQRWDRILQASGMKRAKPSPTSTQTPDQASLHAFA DSLERDLDAPSPMHEGDQTRAS

As used herein the predetermined “N-terminus” to “C terminus” orientation of the N-terminal capping region, the DNA binding domain comprising the repeat TALE monomers and the C-terminal capping region provide structural basis for the organization of different domains in the d-TALEs or polypeptides of the invention.

The entire N-terminal and/or C-terminal capping regions are not necessary to enhance the binding activity of the DNA binding region. Therefore, in certain embodiments, fragments of the N-terminal and/or C-terminal capping regions are included in the TALE polypeptides described herein.

In certain embodiments, the TALE (including TALEs) polypeptides described herein contain a N-terminal capping region fragment that included at least 10, 20, 30, 40, 50, 54, 60, 70, 80, 87, 90, 94, 100, 102, 110, 117, 120, 130, 140, 147, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260 or 270 amino acids of an N-terminal capping region. In certain embodiments, the N-terminal capping region fragment amino acids are of the C-terminus (the DNA-binding region proximal end) of an N-terminal capping region. N-terminal capping region fragments that include the C-terminal 240 amino acids enhance binding activity equal to the full length capping region, while fragments that include the C-terminal 147 amino acids retain greater than 80% of the efficacy of the full length capping region, and fragments that include the C-terminal 117 amino acids retain greater than 50% of the activity of the full-length capping region.

In some embodiments, the TALE polypeptides described herein contain a C-terminal capping region fragment that included at least 6, 10, 20, 30, 37, 40, 50, 60, 68, 70, 80, 90, 100, 110, 120, 127, 130, 140, 150, 155, 160, 170, 180 amino acids of a C-terminal capping region. In certain embodiments, the C-terminal capping region fragment amino acids are of the N-terminus (the DNA-binding region proximal end) of a C-terminal capping region. In certain embodiments, C-terminal capping region fragments that include the C-terminal 68 amino acids enhance binding activity equal to the full length capping region, while fragments that include the C-terminal 20 amino acids retain greater than 50% of the efficacy of the full length capping region.

In certain embodiments, the capping regions of the TALE polypeptides described herein do not need to have identical sequences to the capping region sequences provided herein. Thus, in some embodiments, the capping region of the TALE polypeptides described herein have sequences that are at least 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical or share identity to the capping region amino acid sequences provided herein. Sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. In some preferred embodiments, the capping region of the TALE polypeptides described herein have sequences that are at least 95% identical or share identity to the capping region amino acid sequences provided herein.

Sequence homologies may be generated by any of a number of computer programs known in the art, which include but are not limited to BLAST or FASTA. Suitable computer program for carrying out alignments like the GCG Wisconsin Bestfit package may also be used. Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. % homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

Additional sequences for the conserved portions of polypeptide monomers and for N-terminal and C-terminal capping regions are included in the sequences with the following gene accession numbers: AAW59491.1, AAQ79773.2, YP_450163.1, YP_001912778.1, ZP_02242672.1, AAW59493.1, AAY54170.1, ZP_02245314.1, ZP_02243372.1, AAT46123.1, AAW59492.1, YP_451030.1, YP_001915105.1, ZP_02242534.1, AAW77510.1, ACD11364.1, ZP 02245056.1, ZP 02245055.1, ZP 02242539.1, ZP 02241531.1, ZP 02243779.1, AAN01357.1, ZP_02245177.1, ZP_02243366.1, ZP_02241530.1, AAS58130.3, ZP_02242537.1, YP_200918.1, YP_200770.1, YP_451187.1, YP_451156.1, AAS58127.2, YP_451027.1, UR_451025.1, AAA92974.1, UR_001913755.1, ABB70183.1, UR_451893.1, UR_450167.1, ABY60855.1, UR 200767.1, ZR_02245186.1, ZR_02242931.1, ZR_02242535.1, AAU54169.1, UR_450165.1, UR_001913452.1, AAS58129.3, ACM44927.1, ZR_02244836.1, AAT46125.1, UR_450161.1, ZR_02242546.1, AAT46122.1, UR_451897.1, AAF98343.1, UR_001913484.1, AAY54166.1, UR_001915093.1, UR_001913457.1, ZR_02242538.1, UR_200766.1, UR_453043.1, UR_001915089.1, UR_001912981.1, ZR_02242929.1, UR_001911730.1, UR_201654.1, UR_199877.1, ABB70129.1, UR_451696.1, UR_199876.1, AAS75145.1, AAT46124.1, UR_200914.1, UR_001915101.1, ZR_02242540.1, AAG02079.2, UR_451895.1, YP 451189.1, UR_200915.1, AAS46027.1, UR_001913759.1, UR_001912987.1, AAS58128.2, AAS46026.1, UR_201653.1, UR_202894.1, UR_001913480.1, ZR_02242666.1, R_001912775.1, ZR_02242662.1, AAS46025.1, AAC43587.1, BAA37119.1, NPJ544725.1, AB077779.1, BAA37120.1, ACZ62652.1, BAF46271.1, ACZ62653.1, NPJ544793.1, AB077780.1, ZR_02243740.1, ZR_02242930.1, AAB69865.1, AAY54168.1, ZR_02245191.1, UR_001915097.1, ZR_02241539.1, UR_451158.1, BAA37121.1, UR_001913182.1, UR_200903.1, ZR_02242528.1, ZR_06705357.1, ZR_06706392.1, ADI48328.1, ZR_06731493.1, ADI48327.1, AB077782.1, ZR_06731656.1, NR_942641.1, AAY43360.1, ZR_06730254.1, ACN39605.1, UR_451894.1, UR_201652.1, UR_001965982.1, BAF46269.1, NPJ544708.1, ACN82432.1, AB077781.1, P14727.2, BAF46272.1, AAY43359.1, BAF46270.1, NR_644743.1, ABG37631.1, AAB00675.1, YP 199878.1, ZR_02242536.1, CAA48680.1, ADM80412.1, AAA27592.1, ABG37632.1, ABP97430.1, ZR_06733167.1, AAY43358.1, 2KQ5_A, BAD42396.1, ABO27075.1, UR_002253357.1, UR_002252977.1, ABO27074.1, ABO27067.1, ABO27072.1, ABO27068.1, UR_003750492.1, ABO27073.1, NR_519936.1, ABO27071.1, AB027070.1, and AB027069.1, each of which is hereby incorporated by reference.

In some embodiments, the TALEs described herein also include a nuclear localization signal and/or cellular uptake signal. Such signals are known in the art and may target a TALE to the nucleus and/or intracellular compartment of a cell. Such cellular uptake signals include, but are not limited to, the minimal Tat protein transduction domain which spans residues 47-57 of the human immunodeficiency virus Tat protein: YGRKKRRQRRR.

In some embodiments, the TALEs described herein include a nucleic acid or DNA binding domain that is a non-TALE nucleic acid or a non-TALE DNA binding domain.

As used herein the term “non-TALE DNA binding domain” refers to a DNA binding domain that has a nucleic acid sequence corresponding to a nucleic acid sequence which is not substantially homologous to a nucleic acid that encodes for a TALE protein or fragment thereof, e.g., a nucleic acid sequence which is different from a nucleic acid that encodes for a TALE protein and which is derived from the same or a different organism.

In certain embodiments, the TALEs described herein include a nucleic acid or DNA binding domain that is linked to a non-TALE polypeptide.

A “non-TALE polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to a TALE protein or fragment thereof, e.g., a protein which is different from a TALE protein and which is derived from the same or a different organism. In this context, the term “linked” is intended include any manner by which the nucleic acid binding domain and the non-TALE polypeptide could be connected to each other, including, for example, through peptide bonds by being part of the same polypeptide chain or through other covalent interactions, such as a chemical linker. The non-TALE polypeptide may be linked, for example to the N-terminus and/or C-terminus of the nucleic acid binding domain, may be linked to a C-terminal or N-terminal cap region, or may be connected to the nucleic acid binding domain indirectly.

In certain embodiments, the TALEs or polypeptides of the invention comprise chimeric DNA binding domains. Chimeric DNA binding domains may be generated by fusing a full TALE (including the N- and C-terminal capping regions) with another TALE or non-TALE DNA binding domain such as zinc finger (ZF), helix-loop-helix, or catalytically-inactivated DNA endonucleases (e.g., EcoRI, meganucleases, etc.), or parts of TALE may be fused to other DNA binding domains. The chimeric domain may have novel DNA binding specificity that combines the specificity of both domains.

In certain embodiments, the TALE polypeptides of the invention include a nucleic acid binding domain linked to the one or more effector domains. In certain embodiments, the effector domain is a nickase or nuclease.

In certain embodiments, the sequence-specific nuclease is a zinc finger nuclease (ZFN), such as an artificial zinc-finger nuclease having arrays of zinc-finger (ZF) modules to target new DNA-binding sites in a target sequence (e.g., target sequence or target site in the genome). Each zinc finger module in a ZF array targets three DNA bases. A customized array of individual zinc finger domains is assembled into a ZF protein (ZFP). The resulting ZFP can be linked to a functional domain such as a nuclease.

ZF nucleases (ZFN) may be used as alternative programmable nucleases for use in retron-based editing in place of RNA-guide nucleases. ZFN proteins have been extensively described in the art, for example, in Carroll et al., “Genome Engineering with Zinc-Finger Nucleases,” Genetics, August 2011, Vol. 188: 773-782; Durai et al., “Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells,” Nucleic Acids Res, 2005, Vol. 33: 5978-90; and Gaj et al., “ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering,” Trends Biotechnol. 2013, Vol. 31: 397-405, each of which are incorporated herein by reference in their entireties.

In certain embodiments, the ZF-linked nuclease is a catalytic domain of the Type IIS restriction enzyme FokI (see Kim et al., PNAS U.S.A. 91:883-887, 1994; Kim et al., PNAS U.S.A. 93:1156-1160, 1996, both incorporated herein by reference).

In certain embodiments, the ZFN comprises paired ZFN heterodimers, resulting in increased cleavage specificity and/or decreased off-target activity. In this embodiment, each ZFN in the heterodimer targets different nucleotide sequences separated by a short spacer (see Doyon et al., Nat. Methods 8:74-79, 2011, incorporated herein by reference).

In certain embodiments, the ZFN comprises a polynucleotide-binding domain (comprising multiple sequence-specific ZF modules) and a polynucleotide cleavage nickase domain.

In certain embodiments, the ZFs are engineered using libraries of two finger modules.

In certain embodiments, strings of two-finger units are used in ZFNs to improve DNA binding specificity from polyzinc finger peptides (see PNAS USA 98: 1437-1441, incorporated herein by reference).

In certain embodiments, the ZFN has more than 3 fingers. In certain embodiments, the ZFN has 4, 5, or 6 fingers. In certain embodiments, the ZF modules in the ZFN are separated by one or more linkers to improve specificity.

In certain embodiments, the ZF of the ZFN includes substitutions in the dimer interface of the cleavage domain that prevent homodimerization between ZFs, but allow heterodimers to form.

In certain embodiments, the ZF of the ZFN has a design that retains activity while suppressing homodimerization.

In certain embodiments, the ZFN is any one of the ZF nucleases in Table 1 of Carroll et al., Genetics 188(4):773-782, 2011, incorporated herein by reference.

General principles and guidance for generating ZF, ZF arrays, and ZFN can be found in the art, such as the modular design (where the different modules can be rearranged and assembled into new combinations for new targets) of the ZF or ZF arrays in the ZFN as taught in Carroll et al., Nat. Protoc. 1: 1329-1341, 2006 (incorporated herein by reference); the new three-finger sets for engineered ZFs generated by using partially randomized libraries; profiling the DNA-binding specificities of engineered Cys2His2 zinc finger domains using a rapid cell-based method (see Nucleic Acids Res. 35: e81, incorporated by reference). ZFs for certain DNA triplets that work well in neighbor combination are described in Sander et al., 2011. Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA) is taught in Nat. Methods 8: 67-69). ToolGen describes the individual fingers in their collection that are best behaved in modular assembly (Kim et al., 2011). Preassembled zinc-finger arrays for rapid construction of ZFNs are taught in Nat. Methods 8:7.

Additional, non-limiting ZFs and AFNz that can be adapted for use in the instant invention include those described in WO2010/065123, WO2000/041566, WO2003/080809, WO2015/143046, WO2016/183298, WO2013/044008, WO2015/031619, WO2017/136049, WO2016/014794, WO2017/091512, WO1995/009233, WO2000/023464, WO2000/042219, WO2002/026960, WO2001/083793; U.S. Pat. Nos. 9,428,756, 9,145,565, 8,846,578, 8,524,874, 6,777,185, 6,599,692, 7,235,354, 6,503,717, 7,491,531, 7,943,553, 7,262,054, 8,680,021, 7,705,139, 7,273,923, 6,780,590, 6,785,613, 7,788,044, 7,177,766, 6,453,242, 6,794,136, 7,358,085, 8,383,766, 7,030,215, 7,013,219, 7,361,635, 7,939,327, 8,772,453, 9,163,245, 7,045,304, 8,313,925, 9,260,726, 6,689,558, 8,466,267, 7,253,273, 7,947,873, 9,388,426, 8,153,399, 8,569,253, 8,524,221, 7,951,925, 9,115,409, 8,772,008, 9,121,072, 9,624,498, 6,979,539, 9,491,934, 6,933,113, 9,567,609, 7,070,934, 9,624,509, 8,735,153, 9,567,573, 6,919,204, US2002-0081614, US2004-0203064, US2006-0166263, US2006-0292621, US2003-0134318, US2006-0294617, US2007-0287189, US2007-0065931, US2003-0105593, US2003-0108880, US2009-0305402, US2008-0209587, US2013-0123484, US2004-0091991, US2009-0305977, US2008-0233641, US2014-0287500, US2011-0287512, US2009-0258363, US2013-0244332, US2007-0134796, US2010-0256221, US2005-0267061, US2012-0204282, US2012-0252122, US2010-0311124, US2016-0215298, US2008-0031109, US2014-0017214, US2015-0267205, US2004-0235002, US2004-0204345, US2015-0064789, US2006-0063231, US2011-0265198, US2017-0218349, all incorporated herein by reference.

Polynucleotides and vectors capable of expressing one or more of the ZFNs are also provided herein, which can be part of the vector system of the invention. The polynucleotides and vectors can be expressed in a cell, such as a eukaryotic cell, a mammalian cell, or a human cell. Suitable vectors, cells and expression systems are described in greater detail elsewhere herein, and can be suitable for use with the TALEs, the meganucleases, and the CRISPR-Cas nucleases.

In certain embodiments, the sequence-specific nuclease is a meganuclease.

Meganucleases are a class of sequence-specific endonucleases that recognize large DNA target sites (>12 bp). These proteins can cleave a unique chromosomal sequence without affecting overall genome integrity. Meganucleases create site specific DNA DSBs, and, in the presence of donor DNA, such as one present in the heterologous nucleic acid encompassed by or encoded by the engineered retron of the invention, promotes the integration of the donor DNA at the cleavage site through homologous recombination (HR).

In certain embodiments, the meganuclease is a homing endonuclease, which is a widespread class of proteins found in eukaryotes, bacteria and archaea. In certain embodiments, the meganuclease is of the LAGLIDADG family of homing endonucleases.

In certain embodiments, the meganuclease is I-Scel, I-Cre-I, I-Dmol, or an engineered or a naturally occurring variant thereof. The hallmark of these proteins is a well conserved LAGLIDADG peptide motif, termed (do)decapeptide, found in one or two copies. Homing endonucleases with only one such motif, such as I-Crel or I-Ceul, function as homodimers. In contrast, larger proteins bearing two (do)decapeptide motifs, such as I-Scel, Pl-Scel and I-Dmol are single chain proteins.

Additional homing nucleases are found at the website of homingendonuclease.net, which provides a database listing basic properties of known LAGLIDADG homing endonucleases. See also Taylor et al., Nucleic Acids Research 40 (W1): W110-W116, 2012 (all incorporated herein by reference).

In certain embodiments, specificity (or polynucleotide recognition) of the meganuclease is modified by altering the amino acids within the meganuclease, and/or by fusing other effector domains with the meganuclease.

In certain embodiments, the meganuclease is a megaTAL, which includes a DNA binding domain from a TALE.

In certain embodiments, the meganuclease is engineered to have nickase activity.

Additional suitable natural and engineered meganucleases and megaTALs are described in WO2006/097853, WO2004/067736, WO2012/030747, WO2007/123636, WO2010/001189, WO2018/071565, WO2007/049095, WO2009/068937, WO2005/105989, WO2008/102198, WO2007/057781, WO2019/126558, WO2010/046786, US2010-0151556, US2014-0121115, US2011-0207199, US2012-0301456, US2013-0189759, US2011-0158974, US2010-0144012, US2014-0112904, US2013-0196320, US2010-0203031, US2010-0167357, US2012-0272348, US2012-0258537, US2011-0072527, US2013-0183282, US2014-0178942, US2012-0260356, US2013-0236946, US2010-0325745, US2011-0041194, US2014-0004608, US2011-0263028, US2011-0225664, US2013-0145487, US2013-0045539, US2012-0171191, US2015-0315557, US2014-0017731, US2011-0091441, US2014-0038239, US2010-0229252, US2009-0222937, US2010-0146651, US2013-0059387, US2011-0179507, US2013-0326644, US2006-0078552, US2004-0002092, US2012-0052582, US2009-0162937, US2010-0086533, US2009-0220476, U.S. Pat. Nos. 8,802,437, 7,842,489, 8,715,992, 8,426,177, 8,476,072, 9,365,864, 9,540,623, 9,273,296, 9,290,748, 8,163,514, 8,148,098, 8,143,016, 8,143,015, 8,133,697, 8,129,134, 8,124,369, 8,119,361, 7,897,372, 9,683,257, U.S. Ser. No. 10/287,626, U.S. Ser. No. 10/273,524, U.S. Ser. No. 10/000,746, U.S. Ser. No. 10/006,052, U.S. Pat. Nos. 7,919,605, 9,018,364, U.S. Ser. No. 10/407,672, U.S. Pat. Nos. 8,211,685, 9,365,864, 7,476,500, all incorporated herein by reference.

In certain embodiments, the sequence-specific nuclease is TnpB, which is a programmable RNA-guided DNA endonuclease. It is believed that TnpB is a functional progenitor of the CRISPR-Cas nucleases.

Transposons are mobile genetic elements that contain only the genes required for their transposition and its regulation. These elements encode the tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. TnpB has been shown to be a nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5′-TTGAT transposon-associated motif, and TnpB can be reprogrammed to cleave DNA target sites in human cells.

In certain embodiments, tnpB is from D. radiodurans ISDra2 of the IS200/IS605 family.

In certain embodiments, tnpB is from transposon PsiTn554.

In certain embodiments, the sequence-specific nuclease is a TnpB-like protein, such as Fanzor1 or Fanzor2. These proteins are widespread in diverse eukaryotic transposable elements (TEs), and in large double-stranded DNA (dsDNA) viruses infecting eukaryotes. Fanzor and TnpB proteins share the same conserved amino acid motif in their C-terminal half regions: D-X(125, 275)-[TS]-[TS]-X-X-[C4 zinc finger]-X(5,50)-RD, but are highly variable in their N-terminal regions. Fanzor1 proteins are frequently captured by DNA transposons from different superfamilies including Helitron, Mariner, IS4-like, Sola and MuDr. In contrast, Fanzor2 proteins appear only in some IS607-type elements.

In certain embodiments, the sequence-specific nuclease is IscB.

ISC (insertion sequences Cas9-like) is a novel group of bacterial and archaeal DNA transposons that encode Cas9 homologs. The ISC transposon-encoded two nuclease domain-containing proteins are the likely ancestors of the CRISPR-associated Cas9. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. ISC genes, however, are not linked to Cas genes or CRISPR. They represent a distinct group of nonautonomous transposons, with many diverse families of full-length ISC transposons. Their terminal sequences (particularly 3′ termini) are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene, and often also encode the TnpB protein containing the RuvC-like endonuclease domain. The terminal regions of the ISC and IS605 transposons contain palindromic structures that are likely recognized by the Y1 transposase. The transposons from these two groups are inserted either exactly in the middle or upstream of specific 4-bp target sites, without target site duplication.

In certain embodiments, the sequence-specific nuclease is a restriction endonuclease (RE), such as an RE with stringent/long recognition sequence of at least 8 nts.

In certain embodiments, the RE is a rare-cutter RE with seven and eight base pair recognition sequences. Exemplary rare-cutter RE enzyme include NotI, which cuts after the first GC of a 5′-GCGGCCGC-3′ sequence.

In certain embodiments, the components of the system—e.g., the retron encoded ncRNA or msDNA in complex with the RT, the sequence-specific nuclease, and the DNA-repair modulating biomolecule, may form multiple complexes in a so-called split complex configuration. The multiple complexes may be brought together to form a functional complex.

For example, in some embodiments, a first component in the system may be a split protein or domain. A fragment of the split protein or domain may associate with a second component of the system, while another fragment of the split protein or domain may associate with a third component of the system. The two fragments of the split protein or domain may be brought together (e.g., along with the other components of the system) to form a functional complex.

In certain embodiments, the split protein or domain is the sequence-specific nuclease, e.g., a CRISPR/Cas effector enzyme (e.g., Cas protein such as Cas9 or Cas12), a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE).

In certain embodiments, the split protein or domain is the reverse transcriptase domain.

In certain embodiments, the split protein or domain is the DNA-repair modulating biomolecule.

For example a first fragment of the sequence-specific nuclease may associate with the reverse transcriptase domain and a second fragment of the sequence-specific nuclease may associate with the DNA-repair modulating biomolecule. The two fragments of the split protein or domain may be brought together (e.g., along with the reverse transcriptase domain and the DNA-repair modulating biomolecule) to form a functional complex. The associations between the parts of the split protein or domain may be through adaptor proteins or linkers described herein (e.g., those used for associating Cas proteins with function domains).

In certain embodiments, the split protein or domain is split in the sense that the two parts of the split protein or domain substantially comprise a functioning split protein or domain. Ideally, the split should always be so that the catalytic domain(s) are unaffected. That split protein or domain may function as a sequence-specific nuclease or it may be a dead-Cas which is essentially an RNA-binding protein with very little or no catalytic activity, due to typically mutation(s) in its catalytic domains.

Each fragment of the split protein or domain may be fused to a dimerization partner. For example, rapamycin sensitive dimerization domains enables a chemically inducible split protein or domain for temporal control of the split protein or domain's activity. The split protein or domain can thus be rendered chemically inducible by being split into two fragments and that rapamycin-sensitive dimerization domains may be used for controlled reassembly of the split protein or domain. The two parts of the split protein or domain can be thought of as the N′ terminal part and the C′ terminal part of the split protein or domain. The fusion is typically at the split point of the split protein or domain. In other words, the C′ terminal of the N′ terminal part of the split protein or domain is fused to one of the dimer halves, whilst the N′ terminal of the C′ terminal part is fused to the other dimer half.

The split protein or domain does not have to be split in the sense that the break is newly created. The split point is typically designed in silico and cloned into the constructs. Together, the two parts of the split protein or domain, the N′ terminal and C′ terminal parts, form a full split protein or domain, comprising preferably at least 70% or more of the wildtype amino acids (or nucleotides encoding them), at least 80% or more, at least 90% or more, at least 95% or more, and at least 99% or more of the wildtype amino acids (or nucleotides encoding them). When the two parts are brought together, the desired split protein or domain function is restored or reconstituted. The dimer may be a homodimer or a heterodimer.

In certain embodiments, the protein components of the system—e.g., the RT, the sequence-specific nuclease (a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)), and the DNA-repair modulating biomolecule, may further comprise one or more additional functional domains.

In certain embodiments, the functional domain comprises a nuclear localization signal (NLS). In certain embodiments, one or more C-terminal or N-terminal NLSs are attached. In certain embodiments, a C-terminal NLS is attached for expression and nuclear targeting in eukaryotic cells, e.g., human cells. In certain embodiments, the NLS(s) may be at a location that is not at the C-terminus or N-terminus, for example, the NLS(s) may be between two polypeptides.

Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen; the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS); the c-myc NLS; the hRNPA1 M9 NLS; the NLS of the IBB domain from importin-alpha; the NLS of the myoma T protein; the NLS of human p53; the NLS of mouse c-abl IV; the NLS of the influenza virus NS1; the NLS of the Hepatitis virus delta antigen; the NLS of the mouse Mx1 protein; the NLS of the human poly(ADP-ribose) polymerase; and the NLS of the steroid hormone receptors (human) glucocorticoid. Exemplary NLS sequences include those described in paragraph [00106] of Feng Zhang et al. (WO2016/106236), all incorporated herein by reference.

In certain embodiments, the functional domain comprises at least two NLS domains. The one or more NLS domain(s) may be positioned at or near or in proximity to a terminus of a polypeptide and, if two or more NLSs, each of the two may be positioned at or near or in proximity to a terminus of the polypeptide.

In any of the fusion proteins, the fusion between the two domains (such as the RT and Cas enzyme, or the Cas and the DNA-repair modulating biomolecule) may be linked through a linker.

A “linker” as used herein includes a peptide which joins two proteins or domains to form a fusion protein. Generally, such molecules have no specific biological activity other than to join or to preserve some minimum distance or other spatial relationship between the proteins/domains. However, in certain embodiments, the linker may be selected to influence some property of the linker and/or the fusion protein such as the folding, net charge, or hydrophobicity of the linker. Suitable linkers for use in the present disclosure are well-known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. However, as used herein, the linker may also be a covalent bond (carbon-carbon bond or carbon-heteroatom bond).

In particular embodiments, the linker is used to separate the sequence-specific nuclease (a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)) and the RT and/or the DNA-repair modulating biomolecule by a distance sufficient to ensure that each protein domain retains its required functional property.

Preferred peptide linker sequences adopt a flexible extended conformation and do not exhibit a propensity for developing an ordered secondary structure. In certain embodiments, the linker can be a chemical moiety which can be monomeric, dimeric, multimeric or polymeric. In certain embodiments, the linker comprises amino acids. Typical amino acids in flexible linkers include Gly, Asn and Ser. Accordingly, in particular embodiments, the linker comprises a combination of one or more of Gly, Asn and Ser amino acids. Other near neutral amino acids, such as Thr and Ala, also may be used in the linker sequence.

In certain embodiments, the linker comprises a GlySer-rich sequence, such as a G_(n)S linker (n=1, 2, 3, 4, or 5, such as GS or G4S), or repeats thereof (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 repeats, optionally with an overall length of about 4-30 residues, 4-20 residues, or 4-10 residues).

In certain embodiments, the linker comprises a G4S linker with 3, 6, 9, or 12 repeats.

In certain embodiments, the linker is one disclosed in Maratea et al., Gene 40: 39-46, 1985; Murphy et al., PNAS USA 83: 8258-62, 1986; U.S. Pat. No. 4,935,233; or U.S. Pat. No. 4,751,180, all incorporated by reference.

In certain embodiments, the linker comprises a GlySer linker such as GGS, GGGS, GSG, GGGGS, optionally with repeats of 3 (such as (GGS)₃, (GGGGS)₃), 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more, to provide suitable lengths.

In certain embodiments, the linker comprises (GGGGS)₃₋₁₅, such as (GGGGS)₃₋₁₁, e.g., GGGGS with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 repeats.

In certain embodiments, the linker comprises

LEPGEKPYKCPECGKSFSQSGALTRHQR THTR.

In yet an additional embodiment, the linker is an XTEN linker.

In certain embodiments, N- and/or C-terminal NLSs also function as linker (e.g., PKKKRKVEASSPKKRKVEAS).

The gRNA and the various nucleases and fusions thereof can be provided in the form of a protein, optionally where the nuclease is complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector). In some embodiments, the RNA-guided nuclease and the gRNA are both provided by vectors. Both can be expressed by a single vector or separately on different vectors. The vectors encoding the RNA-guided nuclease and gRNA may be included in the vector system comprising the engineered retron msr gene, msd gene and ret gene sequences.

Codon usage may be optimized to improve production of the engineered retron e.g., retron reverse transcriptase, ncRNA and/or RNA-guided nuclease in a particular cell or organism. For example, a nucleic acid encoding an ncRNA, RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a the particular cell such as a eukaryotic cell (e.g., yeast cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell), or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the reverse transcriptase or ncRNA is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell.

F. RT-PN Fusion Proteins

The recombinant retron-based editing system described herein contemplates fusion proteins comprising a programmable nuclease (PN) and a RT, optionally joined by a linker. The application contemplates any suitable programmable nuclease and RT (e.g., retron RTs of Table A) to be combined in a single fusion protein. In one embodiment, the RT is joined to the N-terminus of the PN. In another embodiment, the RT is joined to the C-terminus of the PN. The Examples of PNs and RTs are each defined herein.

In various embodiments, the fusion proteins may comprise any suitable structural configuration. For example, the fusion protein may comprise from the N-terminus to the C-terminus direction, a PN fused to a RT. In other embodiments, the fusion protein may comprise from the N-terminus to the C-terminus direction, a RT fused to a NP. The fused domain may optionally be joined by a linker, e.g., an amino acid sequence.

G. Nuclear Localization Signals

In various embodiments, any of the polypeptide components of the retron-based editing systems may be engineered with one or more nuclear localization signals, which help promote translocation of a protein into the cell nucleus. The polypeptides of the retron-based editing system may comprise any known NLS sequence, including any of those described in Cokol et al., “Finding nuclear localization signals,” EMBO Rep., 2000, 1(5): 411-415 and Freitas et al., “Mechanisms and Signals for the Nuclear Import of Proteins,” Current Genomics, 2009, 10(8): 550-7, each of which are incorporated herein by reference.

In various embodiments, the polypeptides disclosed herein may include one or more, preferably, at least two nuclear localization signals. The NLSs may be any known NLS sequence in the art. The NLSs may also be any future-discovered NLSs for nuclear localization. The NLSs also may be any naturally-occurring NLS, or any non-naturally occurring NLS (e.g., an NLS with one or more desired mutations). The term“nuclear localization sequence” or “NLS” refers to an amino acid sequence that promotes import of a protein into the cell nucleus, for example, by nuclear transport. Nuclear localization sequences are known in the art and would be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., International PCT application PCT/EP2000/011690, filed Nov. 23, 2000, published as WO/2001/038547 on May 31, 2001, the contents of which are incorporated herein by reference. In some embodiments, an NLS comprises the amino acid sequence PKKKRKV.

A representative nuclear localization signal is a peptide sequence that directs the protein to the nucleus of the cell in which the sequence is expressed. A nuclear localization signal is predominantly basic, can be positioned almost anywhere in a protein's amino acid sequence, generally comprises a short sequence of four amino acids (Autieri & Agrawal, (1998) J. Biol. Chem. 273: 14731-37, incorporated herein by reference) to eight amino acids, and is typically rich in lysine and arginine residues (Magin et al., (2000) Virology 274: 11-16, incorporated herein by reference). Nuclear localization signals often comprise proline residues. A variety of nuclear localization signals have been identified and have been used to effect transport of biological molecules from the cytoplasm to the nucleus of a cell. See, e.g., Tinland et al., (1992) Proc. Natl. Acad. Sci. U.S.A.89:7442-46; Moede et al., (1999) FEBS Lett. 461:229-34, which is incorporated by reference. Translocation is currently thought to involve nuclear pore proteins.

Most NLSs can be classified in three general groups: (i) a monopartite NLS exemplified by the SV40 large T antigen NLS (PKKKRKV); (ii) a bipartite motif consisting of two basic domains separated by a variable number of spacer amino acids and exemplified by the Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL); and (iii) noncanonical sequences such as M9 of the hnRNP A1 protein, the influenza virus nucleoprotein NLS, and the yeast Gal4 protein NLS (Dingwall and Laskey 1991). Nuclear localization signals appear at various points in the amino acid sequences of proteins. NLS's have been identified at the N-terminus, the C-terminus and in the central region of proteins. Thus, the disclosure provides polypeptides that may be modified with one or more NLSs at the C-terminus, the N-terminus, as well as at in internal region of a polypeptide (including a fusion protein).

The present disclosure contemplates any suitable means by which to modify a polypeptide to include one or more NLSs. In one aspect, a polypeptide (e.g., a programmable nuclease) may be engineered to express with a translationally fused NLS at its N-terminus or its C-terminus (or both), i.e., to form a polypeptide-NLS fusion construct. In addition, the NLSs may include various amino acid linkers or spacer regions encoded between a polypeptide and the N-terminally, C-terminally, or internally-attached NLS amino acid sequence, e.g, and in the central region of proteins.

Thus, the present disclosure also provides for nucleotide constructs, vectors, and host cells for expressing fusion proteins that comprise a polypeptide and one or more NLSs.

H. DNA-Repair Modulating Biomolecules

In certain embodiments, the engineered retron described herein (e.g., an engineered nucleic acid construct or engineered nucleic acid-enzyme construct described herein) further comprises or encodes a DNA-repair modulating biomolecule, which may further enhance the efficiency of integration of a transgene on the heterologous nucleic acid by homology dependent repair (HDR).

In certain embodiments, the DNA-repair modulating biomolecule comprises a Nonhomologous end joining (NHEJ) inhibitor.

In certain embodiments, the DNA-repair modulating biomolecule comprises a homologous directed repair (HDR) promoter.

In certain embodiments, the DNA-repair modulating biomolecule comprises a NHEJ inhibitor and an HDR promoter.

In certain embodiments, the DNA-repair modulating biomolecule enhances or improves more precise genome editing and/or the efficiency of homologous recombination, compared to the otherwise identical embodiment without the DNA-repair modulating biomolecule.

HDR promoters and/or NHEJ inhibitors can, in some embodiments, comprise one or more small molecules. Systems bearing recombination enhancers such as small molecules that activate HDR and suppress NHEJ locally at the genomic site of the DNA damage can be tailored in their placement on the engineered systems to further enhance their efficiency. In general, the small molecule recombination enhancers can be synthesized to bear linkers and a functional group, such as maleimide for reacting with a thiol group on a Cys residue of a protein, for chemical conjugation to the engineered systems. Use of commercially available functionalized PEG linkers (alkyne, azide, cyclooctyne etc.) can also be employed for conjugation, and orthogonal conjugation chemistries can be utilized for the multivalent display.

Conjugation sites can be readily identified where modifications do not affect the potency of the recombination enhancers selected.

In certain embodiments, multivalent display of one or more DNA-repair modulating biomolecule can be affected, including multiple moieties of NHEJ inhibitors, HDR promoters, or a combination thereof. See, for example, “Genomic targeting of epigenetic probes using a chemically tailored Cas9 system” by Liszczak et al., Proc Natl Acad Sci U.S.A. 114: 681-686, 2017 (incorporated herein by reference). In certain embodiments, multivalent display of small molecule compounds can be achieved through sortase loop proteins used as a scaffold for their display.

In some embodiments, the DNA-repair modulating biomolecule may comprise an HDR promoter. The HDR promoter may comprise small molecules, such as RSI or analogs thereof. In certain embodiments, the HDR promoter stimulates RAD51 activity or RAD52 motif protein 1 (RDMl) activity. In certain embodiments, the HDR promoter comprises Nocodazole, which can result in higher HDR selection.

In certain embodiments, the HDR promoter may be administered prior to the delivery of the engineered retron described herein.

In certain embodiments, the HDR promoter locally enhances HDR without NHEJ inhibition. For example, RAD5l is a protein involved in strand exchange and the search for homology regions during HDR repair. In certain embodiments, the HDR promoter is phenylbenzamide RSI, identified as a small-molecule RAD51-stimulator (see WO2019/135816 at [0200]-[0204], specifically incorporated herein by reference).

In certain embodiments, the DNA-repair modulating biomolecule comprises C-terminal binding protein interacting protein (CtIP) or a functional fragment or homolog thereof. CtIP is a key protein in early steps of homologous recombination. According to this embodiment, the CtIP or the functional fragment or homolog thereof can be linked (e.g., fused) to the RT or the sequence-specific nuclease (e.g., a CRISPR/Cas effector enzyme, a ZFN, a TALEN, a meganuclease, TnpB, IscB, or a restriction endonuclease (RE)), and stimulates transgene integration by HDR.

In certain embodiments, the CtIP fragment is a minimal N-terminal fragment of the wild-type CtIP, such as the N-terminal fragment comprising residues 1-296 of the full-length CtIP (the HE for HDR enhancer), as described in Charpentier et al. (Nature Comm., DOI: 10.1038/s41467-018-03475-7, incorporated herein by reference), shown to be sufficient to stimulate HDR. The activity of the fragment depends on CDK phosphorylation sites (e.g., S233, T245, and S276) and the multimerization domain essential for CtIP activity in homologous recombination. Thus alternative fragments comprising the CDK phosphorylation sites and the multimerization domain essential for CtIP activity are also within the scope of the invention.

In certain embodiments, the DNA-repair modulating biomolecule comprises a dominant negative 53BP1.

In certain embodiments, the DNA-repair modulating biomolecule comprises a cell cycle-specific degradation tag, such as the degradation domain of the (human) Geminin, and the (murine) CyclinB2.

In certain embodiments, the DNA-repair modulating biomolecule comprises CyclinB2, a member of the B-type cyclins that associate with p34cdc2, and an essential component of the cell cycle regulatory machinery. CRISPR-mediated knock-in efficiency may be increased by promoting the relative increase in Cas9 activity in G2 phase of the cell cycle, when HDR is more active. In certain embodiments, the degradation domains of the (human) Geminin and (murine) CyclinB2 can be used as either N- or C-terminal fusion (e.g., fusions with a Cas, such as Cas9, or the retron RT) to serve as the DNA-repair modulating biomolecule. These domains are known to determine a cell-cycle specific profile of chimeric proteins, namely an increase in their relative concentration in S and G2 compared to G1, high-jacking the conventional CyclinB2 and Geminin degradation pathways. This produces active Geminin-Cas9 and CyclinB2-Cas9 chimeric proteins, which are degraded in a cell-cycle-dependent manner. Such chimeras shift the repair of the DSBs to the HDR repair pathway compared to the commonly used Cas9.

While not wishing to be bound by particular theory, it is believed that the application of such cell cycle-specific degradation tags permits/promotes more efficient/secure gene editing.

In certain embodiments, the DNA-repair modulating biomolecule comprises a Rad family member protein, such as Rad50, Rad51, Rad52, etc., which functions to promote foreign DNA integration into a host chromosome. Specifically, Rad52 is an important homologous recombinant protein, and its complex with Rad51 plays a key role in HDR, mainly involved in the regulation of foreign DNA in eukaryotes. Key steps in the process of HR include repair mediated by Rad51 and strand exchange. Co-expression of Rad52 as a DNA-repair modulating biomolecule significantly enhances the likelihood of HDR by, e.g., three-fold.

In certain embodiments, the DNA-repair modulating biomolecule comprises a RAD52 protein as, e.g., either an N- or a C-terminal fusion.

In certain embodiments, the DNA-repair modulating biomolecule comprises a RAD52 motif protein 1 (RDM1) that functions similarly as RAD52. RDM1 has been shown to be able to repair DSBs caused by DNA replication, prevent G2 or M cell cycle arrest, and improve HDR selection.

In certain embodiments, the DNA-repair modulating biomolecule comprises a dominant negative version of the tumor suppressor p53-binding protein 1 (53BP1). The wild-type protein 53BP1 is a key regulator of the choice between NHEJ and HDR—it is a pro-NHEJ factor which limits HDR by blocking DNA end resection, and also by inhibiting BRCA1 recruitment to DSB sites. It has been shown that global inhibition of 53BP1 by a ubiquitin variant significantly improves Cas9-mediated HDR frequency in non-hematopoietic and hematopoietic cells with single-strand oligonucleotide delivery or double-strand donor in AAV.

In certain embodiments, the dominant negative (DN) version of the 53BP1 comprises the minimal focus forming region, but lacks domains outside this region, e.g., towards the N-terminus and tandem C-terminal BRCT repeats that recruit key effectors involved in NHEJ, such as RIFI-PTIP and EXPAND, respectively. The 53BP1 adapter protein is recruited to specific histone marks at sites of DSBs via this minimal focus forming region, which comprises several conserved domains including an oligomerization domain (OD), a glycine-arginine rich (GAR) motif, a Tudor domain, and an adjacent ubiquitin-dependent recruitment (UDR) motif. The Tudor domain mediates interactions with histone H4 dimethylated at K2023.

In certain embodiments, a dominant negative version of 53BP1 (DN1S) suppresses the accumulation of endogenous 53BP1 and downstream NHEJ proteins at sites of DNA damage, while upregulating the recruitment of the BRCA1 HDR protein. Such a DN version of the 53BP1 can be used as the DNA-repair modulating biomolecule, either as an N- or a C-terminal fusion (such as a Cas9 fusion, to locally inhibit NHEJ at the Cas9-target site defined by its gRNA, while promoting an increase in HDR, and does not globally affect NHEJ, thereby improving cell viability).

In certain embodiments, the DNA-repair modulating biomolecule comprises an NHEJ inhibitor, such as an inhibitor of DNA ligase IV, a KU inhibitor (e.g., KU70 or KU80), a DNA-PKc inhibitor, or an artemis inhibitor.

In certain embodiments, the NHEJ inhibitor inhibits the NHEJ pathway, enhances HDR, or modulates both. In certain embodiments, the NHEJ inhibitor is a small molecule inhibitor.

In certain embodiments, the small molecule inhibitor of the NHEJ pathway comprises an SCR7 analog, for example, PK66, PK76, PK409.

In certain embodiments, the NHEJ inhibitor comprises a KU inhibitor, for example, KU5788, and KU0060648.

In certain embodiments, a small molecule NHEJ inhibitor is linked to a polyglycine tripeptide through PEG for sortase-mediated ligation, as described in WO2019/135816, Guimaraes et al., Nat Protoc 8:1787-99, 2013; Theile et al., Nat Protoc 8:1800-7, 2013; and Schmohl et al., Curr Opin Chem Biol 22:122-8, 2014 (all incorporated herein by reference). The same means can also be used for attaching small molecule HDR enhancers to protein.

An exemplary method for conjugating a small molecule DNA-repair modulating biomolecule without loss of activity is described in WO2019135816, where SCR-7 conjugation of a poly-glycine peptide with the para-carboxylic moiety at ring 4 retained activity of the inhibitor, with rings 1, 2 and 3 of the molecule having involvement in the target-engagement, providing a simple and effective strategy to ligate a small molecule NHEJ inhibitor to the system described herein (e.g., to the sequence-specific nuclease including Cas enzymes, or to the RT) to precisely enhance HDR pathway near a nucleic acid target site.

In certain embodiments, a nucleic acid targeting moiety conjugates based on small molecule inhibitor of DNA-dependent protein kinase (DNA-PK) or heterodimeric Ku (KU70/KU80) can be utilized. KU-0060648 is one potent KU-inhibitors, which can also be functionalized with poly-glycine and used for recombination enhancement.

In certain embodiments, the DNA-repair modulating biomolecule comprises the Tumor Suppressor p53. p53 plays a direct role in DNA repair, including HR regulation, where it affects the extension of new DNA, thereby affecting HDR selection. In vivo, p53 binds to the nuclear matrix and is a rate-limiting factor in repairing DNA structure. p53 regulates DNA repair processes in almost all eukaryotes via transactivation-dependent and -independent pathways, but only the transactivation-independent function of p53 is involved in HR regulation. Wild-type p53 protein can link double stranded breaks to form intact DNA, as well as also playing a role in inhibiting NHEJ. p53 interacts with HR-related proteins, including Rad51, where it controls HR through direct interaction with Rad51.

I. Vectors for Expression of Engineered Retrons

Delivery of an engineered retron to a cell can generally be accomplished with or without vectors. Delivery of the ncRNA encoded by the engineered retron generally does not require a vector used to produce the ncRNA from the engineered retron. For example, the ncRNA can be packaged directly into a delivery vehicle such as a lipid nanoparticle and delivered into a host cell, as described in other sections.

The engineered retrons (or vectors containing them) may be introduced into any type of cell, including any cell from a prokaryotic, eukaryotic, or archaeon organism, including bacteria, archaea, fungi, protists, plants (e.g., monocotyledonous and dicotyledonous plants); and animals (e.g., vertebrates and invertebrates). Examples of animals that may be transfected with an engineered retron include, without limitation, vertebrates such as fish, birds, mammals (e.g., human and non-human primates, farm animals, pets, and laboratory animals), reptiles, and amphibians.

The engineered retrons can be introduced into a single cell or a population of cells. Cells from tissues, organs, and biopsies, as well as recombinant cells, genetically modified cells, cells from cell lines cultured in vitro, and artificial cells (e.g., nanoparticles, liposomes, polymersomes, or microcapsules encapsulating nucleic acids) may all be transfected with the engineered retrons.

The engineered retrons can be introduced into cellular fragments, cell components, or organelles (e.g., mitochondria in animal and plant cells, plastids (e.g., chloroplasts) in plant cells and algae).

Cells may be cultured or expanded after transfection with the engineered retron.

Methods of introducing nucleic acids into a host cell are well known in the art. Commonly used methods include chemically induced transformation, typically using divalent cations (e.g., CaCl₂)), dextran-mediated transfection, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, electroporation, protoplast fusion, encapsulation of nucleic acids in liposomes, and direct microinjection of the nucleic acids comprising engineered retrons into nuclei. See, e.g., Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197; herein incorporated by reference in their entireties.

Methods for genetic transformation of plant cells are known in the art and include those set forth in US2022/0145296, and U.S. Pat. Nos. 8,575,425; 7,692,068; 8,802,934; 7,541,517; each of which is herein incorporated by reference in its entirety. See, also, Rakoczy-Trojanowska, M. (2002) Cell Mol Biol Lett. 7:849-858; Jones et al. (2005) Plant Methods 1:5; Rivera et al. (2012) Physics of Life Reviews 9:308-345; Bartlett et al. (2008) Plant Methods 4:1-12; Bates, G. W. (1999) Methods in Molecular Biology 111:359-366; Binns and Thomashow (1988) Annual Reviews in Microbiology 42:575-606; Christou, P. (1992) The Plant Journal 2:275-281; Christou, P. (1995) Euphytica 85:13-27; Tzfira et al. (2004) TRENDS in Genetics 20:375-383; Yao et al. (2006) Journal of Experimental Botany 57:3737-3746; Zupan and Zambryski (1995) Plant Physiology 107:1041-1047; and Jones et al. (2005) Plant Methods 1:5.

The plant cells that have been transformed may be grown into a transgenic organism, such as a plant, in accordance with conventional methods. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84.

Plant material that may be transformed with the engineered retrons described herein includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruit, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. Progeny, variants, and mutants of the regenerated plants are also included within the scope of the disclosure, provided that these parts comprise the genetic modification introduced by the engineered retron. Further provided is a processed plant product or byproduct that retains the genetic modification introduced by the engineered retron.

The engineered retrons described herein may be used to produce transgenic plants with desired phenotypes, including but not limited to, increased disease resistance (e.g., increased viral, bacterial of fungal resistance), increased insect resistance, increased drought resistance, increased yield, and altered fruit ripening characteristics, sugar and oil composition, and color.

In some embodiments, the retron msr gene, msd gene, and/or ret gene are expressed in vitro from a vector, such as in an in vitro transcription system. The resulting ncRNA or msDNA can be isolated before being packaged and/or formulated for direct delivery into a host cell. For example, the isolated ncRNA or msDNA can be packaged/formulated in a delivery vehicle such as lipid nanoparticles as described in other sections.

In some embodiments, the retron msr gene, msd gene, and/or ret gene are expressed in vivo from a vector within a cell. The retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.

In other embodiments, the retron msr gene, msd gene, and/or ret gene, and any other components of the retron-based genome editing systems described herein (e.g., guide RNA in trans, programmable nuclease (e.g., in trans)) may be expressed in vivo from RNA delivered to the cell. The retron msr gene, msd gene, and/or ret gene can be introduced into a cell with a single vector or in multiple separate vectors to produce msDNA in a host subject.

Vectors and/or nucleic acid molecules encoding the recombinant retron-based genome editing system or components thereof can include control elements operably linked to the retron sequences, which allow for the production of msDNA either in vitro, or in vivo in the subject species. For example, the retron msr gene, msd gene, and/or ret gene can be operably linked to a promoter to allow expression of the retron reverse transcriptase and/or the msDNA product. In some embodiments, heterologous sequences encoding desired products of interest (e.g., polynucleotide encoding polypeptide or regulatory RNA, donor polynucleotide for gene editing, or protospacer DNA for molecular recording) may be inserted in the msr gene and/or msd gene.

Any eukaryotic, archaeal, or prokaryotic cell, capable of being transfected with a vector or retron delivery system comprising the engineered retron sequences, may be used to produce the msDNA in vivo. The ability of constructs to produce the msDNA along with other retron-encoded products can be empirically determined. For example the transfected cell can be assayed either through phenotypic changes that occur due to the introduced sequences or by direct DNA sequencing.

In some embodiments, the engineered retron is produced by a vector system comprising one or more vectors. In the vector system, the msr gene, the msd gene, and/or the ret gene may be provided by the same vector (i.e., cis arrangement of all such retron elements), wherein the vector comprises a promoter operably linked to the msr gene and/or the msd gene. In some embodiments, the promoter is further operably linked to the ret gene. In other embodiments, the vector further comprises a second promoter operably linked to the ret gene. Alternatively, the ret gene may be provided by a second vector that does not include the msr gene and/or the msd gene (i.e., trans arrangement of msr-msd and ret). In yet other embodiments, the msr gene, the msd gene, and the ret gene are each provided by different vectors (i.e., trans arrangement of all retron elements).

Numerous vectors are available for use in the vector or vector system, including but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.

Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus (AAV) vectors, retroviral vectors, lentiviral vectors, and the like. An expression construct can be replicated in a living cell, or it can be made synthetically.

In some embodiments, the nucleic acid comprising an engineered retron sequence is under transcriptional control of a promoter. In some embodiments, the promoter is competent for initiating transcription of an operably linked coding sequence by a RNA polymerase I, II, or III.

Exemplary promoters for mammalian cell expression include the SV40 early promoter, a CMV promoter such as the CMV immediate early promoter (see, U.S. Pat. Nos. 5,168,062 and 5,385,839, incorporated herein by reference in their entireties), the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the herpes simplex virus promoter, among others. Other nonviral promoters, such as a promoter derived from the murine metallothionein gene, will also find use for mammalian expression.

Exemplary promoters for plant cell expression include the CaMV 35S promoter (Odell et al., 1985, Nature 313:810-812); the rice actin promoter (McElroy et al., 1990, Plant Cell 2:163-171); the ubiquitin promoter (Christensen et al., 1989, Plant Mol. Biol. 12:619-632; and Christensen et al., 1992, Plant Mol. Biol. 18:675-689); the pEMU promoter (Last et al., 1991, Theor. Appl. Genet. 81:581-588); and the MAS promoter (Velten et al., 1984, EMBO J. 3:2723-2730).

In additional embodiments, the retron-based vectors may also comprise tissue-specific promoters to start expression only after it is delivered into a specific tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-b promoter, Mb promoter, Nphs1 promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

These and other promoters can be obtained from or incorporated into commercially available plasmids, using techniques well known in the art. See, e.g., Sambrook et al., supra.

In some embodiments, one or more enhancer elements is/are used in association with the promoter to increase expression levels of the constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMBOJ (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777, and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence. All such sequences are incorporated herein by reference.

In one embodiment, an expression vector for expressing an engineered retron, including the msr gene, msd gene, and/or ret gene comprises a promoter operably linked to a polynucleotide encoding the msr gene, msd gene, and/or ret gene.

In some embodiments, the vector or vector system also comprises a transcription terminator/polyadenylation signal. Examples of such sequences include, but are not limited to, those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence (see, e.g., U.S. Pat. No. 5,122,458).

Additionally, 5′-UTR sequences can be placed adjacent to the coding sequence to further enhance the expression. Such sequences may include UTRs comprising an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a vector. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298: Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (199722 ISO-161)c. A multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. Virol. (1989) 63:1651-1660). the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(251:15125-151301)). an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J Biol. Chem. (2004) 279(51):3389-33971) and the like. A variety of nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17): 7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074-1083), and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363(Pt 1):37-44).

These elements are commercially available in plasmids sold, e.g., by Clontech (Mountain View, Calif.), Invivogen (San Diego, Calif.), Addgene (Cambridge, Mass.) and GeneCopoeia (Rockville, Md.). See also IRESite: The database of experimentally verified IRES structures (iresite.org). An IRES sequence may be included in a vector, for example, to express multiple bacteriophage recombination proteins for recombineering or an RNA-guided nuclease (e.g., Cas9) for HDR in combination with a retron reverse transcriptase from an expression cassette.

In some embodiments, a polynucleotide encoding a viral self-cleaving 2A peptide, such as a T2A peptide, can be used to allow production of multiple protein products (e.g., Cas9, bacteriophage recombination proteins, retron reverse transcriptase) from a single vector or a single transcription unit under one promoter. One or more 2A linker peptides can be inserted between the coding sequences in the multicistronic construct. The 2A peptide, which is self-cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels. 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Jhosea asigna virus and porcine teschovirus-1. See, e.g., Kim et al. (2011) PLoS One 6(4): e18556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10): 625-629, Furler et al. (2001) Gene Ther. 8(11):864-873; herein incorporated by reference in their entireties.

In some embodiments, the expression construct comprises a plasmid suitable for transforming a bacterial host. Numerous bacterial expression vectors are known to those of skill in the art, and the selection of an appropriate vector is a matter of choice. Bacterial expression vectors include, but are not limited to, pACYC177, pASK75, pBAD, pBADM, pBAT, pCal, pET, pETM, pGAT, pGEX, pHAT, pKK223, pMal, pProEx, pQE, and pZA31 Bacterial plasmids may contain antibiotic selection markers (e.g., ampicillin, kanamycin, erythromycin, carbenicillin, streptomycin, or tetracycline resistance), a lacZ gene (b-galactosidase produces blue pigment from x-gal substrate), fluorescent markers (e.g., GFP. mCherry), or other markers for selection of transformed bacteria. See, e.g., Sambrook et al., supra.

In other embodiments, the expression construct comprises a plasmid suitable for transforming a yeast cell. Yeast expression plasmids typically contain a yeast-specific origin of replication (ORI) and nutritional selection markers (e.g., HIS3, URA3, LYS2, LEU2, TRP1, METIS, ura4+, leu1+, ade6+), antibiotic selection markers (e.g., kanamycin resistance), fluorescent markers (e.g., mCherry), or other markers for selection of transformed yeast cells. The yeast plasmid may further contain components to allow shuttling between a bacterial host (e.g., E coif) and yeast cells. A number of different types of yeast plasmids are available including yeast integrating plasmids (Yip), which lack an ORI and are integrated into host chromosomes by homologous recombination; yeast replicating plasmids (YRp), which contain an autonomously replicating sequence (ARS) and can replicate independently; yeast centromere plasmids (YCp), which are low copy vectors containing a part of an ARS and part of a centromere sequence (CEN); and yeast episomal plasmids (YEp), which are high copy number plasmids comprising a fragment from a 2 micron circle (a natural yeast plasmid) that allows for 50 or more copies to be stably propagated per cell.

In other embodiments, the expression construct does not comprise a plasmid suitable for transforming a yeast cell.

In other embodiments, the expression construct comprises a virus or engineered construct derived from a viral genome. A number of viral based systems have been developed for gene transfer into mammalian cells. These include adenoviruses, retroviruses (g-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see e.g., Wamock et al. (2011) Methods Mol. Biol. 737:1-25; Walther et al. (2000) Drugs 60(2):249-271; and Lundstrom (2003) Trends Biotechnol. 21(3): 117-122; herein incorporated by reference in their entireties). The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genomes and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells.

For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Bums et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr. Pharm. Des. 17(24): 2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al. (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2): 132-159; herein incorporated by reference).

A number of adenoviral vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis.

Additionally, various adeno-associated vims (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor LaboratoryPress); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

Another vector system useful for delivering nucleic acids encoding the engineered retrons is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference).

Other viral vectors include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing a nucleic acid molecule of interest (e.g., engineered retron) can be constructed as follows. The DNA encoding the particular nucleic acid sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the sequences of interest into the viral genome. The resulting TK-recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.

In some embodiments, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the nucleic acid molecules of interest. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

Members of the alphavirus genus, such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the polynucleotides of the present invention. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as, Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Pat. No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference. Particularly preferred are chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; herein incorporated by reference in their entireties.

A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the nucleic acids of interest (e.g., engineered retron) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the nucleic acid of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA. The method provides for high level, transient, cytoplasmic production of large quantities of RNA. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

In other approaches to infection with vaccinia or avipox virus recombinants, or to the delivery of nucleic acids using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more templates. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No. 5,135,855.

Insect cell expression systems, such as baculovirus systems, can also be used and are known to those of skill in the art and described in, e.g., Baculovirus and Insect Cell Expression Protocols (Methods in Molecular Biology, D. W. Murhammer ed., Humana Press, 2nd edition, 2007) and L. King The Baculovirus Expression System: A laboratory guide (Springer, 1992). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Thermo Fisher Scientific (Waltham, Mass.) and Clontech (Mountain View, Calif.).

Plant expression systems can also be used for transforming plant cells. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes. For a description of such systems see, e.g., Porta et al., Mol. Biotech. (1996) 5:209-221; and Hackland et al., Arch. Virol. (1994) 139:1-22.

To obtain expression of the engineered retron or the ncRNA encoded thereby, the expression construct or the ncRNA must be delivered into a cell. This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states. One mechanism for delivery is via viral infection where the expression construct is encapsulated in an infectious viral particle.

Several non-viral methods for the transfer of expression constructs into cultured cells also are contemplated. These include the use of calcium phosphate precipitation, DEAE-dextran, electroporation, direct microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high velocity microprojectiles, and receptor-mediated transfection (see, e.g., Graham and Van Der Eb (1973) Virology 52:456-467; Chen and Okayama (1987) Mol. Cell Biol. 7:2745-2752; Rippe et al. (1990) Mol. Cell Biol. 10:689-695; Gopal (1985) Mol. Cell Biol. 5:1188-1190; Tur-Kaspa et al. (1986) Mol. Cell. Biol. 6:716-718; Potter et al. (1984) Proc. Natl. Acad. Sci. USA 81:7161-7165); Harland and Weintraub (1985) J. Cell Biol. 101:1094-1099); Nicolau & Sene (1982) Biochim. Biophys. Acta 721:185-190; Fraley et al. (1979) Proc. Natl. Acad. Sci. USA 76:3348-3352; Fechheimer et al. (1987) Proc Natl. Acad. Sci. USA 84:8463-8467; Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572; Wu and Wu (1987) J. Biol. Chem. 262:4429-4432; Wu and Wu (1988) Biochemistry 27:887-892; herein incorporated by reference). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

Once the expression construct has been delivered into the cell the nucleic acid comprising the engineered retron sequence may be positioned and expressed at different sites. In some embodiments, the nucleic acid comprising the engineered retron sequence may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation). In yet further embodiments, the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or episomes encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.

In some embodiments, the expression construct may simply consist of naked recombinant DNA or plasmids comprising the engineered retron. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well. Dubensky et al. (Proc. Natl. Acad. Sci. USA (1984) 81:7529-7533) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty & Neshif (Proc. Natl. Acad. Sci. USA (1986) 83:9551-9555) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding an engineered retron of interest may also be transferred in a similar manner in vivo and express retron products.

In still another embodiment, a naked DNA expression construct may be transferred into cells by particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al. (1987) Nature 327:70-73). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al. (1990) Proc. Natl. Acad. Sci. USA 87:9568-9572). The microprojectiles may consist of biologically inert substances, such as tungsten or gold beads.

In a further embodiment, the expression construct may be delivered using liposomes. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh & Bachhawat (1991) Liver Diseases, Targeted Diagnosis and Therapy Using Specific Receptors and Ligands, Wu et al. (Eds.), Marcel Dekker, NY, 87-104). Also contemplated is the use of lipofectamine-DNA complexes.

In some embodiments, the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al. (1989) Science 243:375-378). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-I) (Kato et al. (1991) J. Biol. Chem. 266(6):3361-3364).

In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-I. Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.

Other expression constructs which can be employed to deliver a nucleic acid into cells are receptor-mediated delivery vehicles. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu (1993) Adv. Drug Delivery Rev. 12:159-167). Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent. Several ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) and transferrin (see, e.g., Wu and Wu (1987), supra; Wagner et al. (1990) Proc. Natl. Acad. Sci. USA 87(9):3410-3414). A synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al. (1993) FASEB J. 7:1081-1091; Perales et al. (1994) Proc. Natl. Acad. Sci. USA 91(9):4086-4090), and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and a liposome. For example, Nicolau et al. (Methods Enzymol. (1987) 149:157-176) employed lactosy 1-ceramide, a galactose-terminal asialoganglioside, incorporated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes. Thus, it is feasible that a nucleic acid encoding a particular gene also may be specifically delivered into a cell by any number of receptor-ligand systems with or without liposomes. Also, antibodies to surface antigens on cells can similarly be used as targeting moieties.

In some embodiments, the promoters that may be used in the retron delivery systems described herein may be constitutive, inducible, or tissue-specific. In some embodiments, the promoters may be a constitutive promoters. Non-limiting exemplary constitutive promoters include cytomegalovirus immediate early promoter (CMV), simian virus (SV40) promoter, adenovirus major late (MLP) promoter, Rous sarcoma virus (RSV) promoter, mouse mammary tumor virus (MMTV) promoter, phosphoglycerate kinase (PGK) promoter, elongation factor-alpha (EFla) promoter, ubiquitin promoters, actin promoters, tubulin promoters, immunoglobulin promoters, a functional fragment thereof, or a combination of any of the foregoing. In some embodiments, the promoter may be a CMV promoter. In some embodiments, the promoter may be a truncated CMV promoter. In other embodiments, the promoter may be an EFla promoter. In some embodiments, the promoter may be an inducible promoter. Non-limiting exemplary inducible promoters include those inducible by heat shock, light, chemicals, peptides, metals, steroids, antibiotics, or alcohol. In some embodiments, the inducible promoter may be one that has a low basal (non-induced) expression level, such as, e.g., the Tet-On® promoter (Clontech). In some embodiments, the promoter may be a tissue-specific promoter. In some embodiments, the tissue-specific promoter is exclusively or predominantly expressed in liver tissue. Non-limiting exemplary tissue-specific promoters include B29 promoter, CD14 promoter, CD43 promoter, CD45 promoter, CD68 promoter, desmin promoter, elastase-1 promoter, endoglin promoter, fibronectin promoter, Flt-1 promoter, GFAP promoter, GPIIb promoter, ICAM-2 promoter, INF-b promoter, Mb promoter, Nphs1 promoter, OG-2 promoter, SP-B promoter, SYN1 promoter, and WASP promoter.

J. Delivery Systems and Methods of Delivery

The engineered retrons can be delivered by any known delivery system such as those described above. Non-limiting examples of delivery vehicles include lipid particles (e.g. Lipid nanoparticles (LNPs)), non-lipid nanoparticles, exosomes, liposomes, micelles, viral particles, Stable nucleic-acid-lipid particles (SNALPs), lipoplexes/polyplexes, DNA nanoclews, Gold nanoparticles, iTOP, Streptolysin O (SLO), multifunctional envelope-type nanodevice (MEND), lipid-coated mesoporous silica particles, inorganic nanoparticles, and polymeric delivery technology (e.g., polymer-based particles).

All-RNA Delivery System and RNA Ratios

In various embodiments, the retron editing systems disclosed herein may be delivered in an “all-RNA” format. As used herein, the term “all-RNA” format refers to the fact that each of the components of a retron editing system (e.g., the retron RT, the programmable nuclease, the sgRNA, and the ncRNA) are delivered and/or administered as RNA. In some embodiments, the RNA components may be delivered to cells and/or tissues by direct means, such as electroporation or transfection. In other embodiments, the RNA components may be delivered to cells and/or tissues by way of a delivery vehicle, such as an LNP or liposome.

In various embodiments, the retron editing systems described herein may comprise a coding RNA (e.g., linear or circular mRNA) that encodes a retron reverse transcriptase (e.g., any RT from Table X or Table A), a coding RNA (e.g., linear or circular mRNA) that encodes a programmable nuclease, a retron ncRNA (e.g., a ncRNA from Table B), and a guide RNA.

In some embodiments, RT and nuclease components may be encoded on the same coding RNA molecule. The proteins may also be expressed from separate coding RNA molecules. In still other embodiments, the RT and the nuclease components can be fused together as a singular fusion polypeptide having an RT domain and a nuclease domain optionally joined by a linker.

In addition, in some embodiments, the ncRNA and the guide RNA may be fused together as a single RNA molecule. For example, the guide RNA may be located at the 5′ end of the ncRNA. In other embodiments, the guide RNA may be located at the 3′ end of the ncRNA. In some embodiments, the ncRNA may comprise a guide RNA at both the 3′ and the 5′ ends of the ncRNA.

In still other embodiments, the ncRNA and the guide RNA may be separate molecules, i.e., delivered separately.

In still other embodiments, the retron editing system may include both a ncRNA-guide RNA fusion and an additional guide RNA provided as a separate molecule.

In various embodiments, the different RNA components of the all-RNA retron editing system can be combined and administered (e.g., directly or within a delivery vehicle) in different ratios. In some embodiments, the ratios of such RNA components or species can be expressed as molar ratios.

For example, the molar ratio of RT coding RNA to nuclease coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In another example, the molar ratio of nuclease coding RNA to RT coding RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of ncRNA or ncRNA-guide RNA fusion to separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of separate guide RNA to ncRNA or ncRNA-guide RNA fusion can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of ncRNA to separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of separate guide RNA to ncRNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of a coding RNA (e.g., encoding RT and/or nuclease) to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of a coding RNA encoding a retron RT to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of a coding RNA encoding a programmable nuclease to ncRNA or ncRNA-guide RNA fusion, as the case may be, can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of a coding RNA encoding a retron RT or a nuclease to a separate guide RNA can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In still another example, the molar ratio of a separate guide RNA to a coding RNA encoding a retron RT or a nuclease can be about 1:1, about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40.

In certain embodiments, the amount of ncRNA-sgRNA relative to RT mRNA is augmented. In certain embodiments the RT mRNA: ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. In certain embodiments, an RT-Cas9 (or Cas9-RT) fusion is encoded by an mRNA. In certain embodiments, the RT-Cas9 mRNA: ncRNA-sgRNA ratio is about 1:1.5, about 1:2, about 1:2.5, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12, about 1:15, about 1:20. Useful ranges include from 1:1 to 1:2, from 1:1.5 to 1:4, from 1:2 to 1:4, from 1:2 to 1:8, from 1:2 to 1:10, from 1:3 to 1:9, from 1:3 to 1:12, from 1:3 to 1:15, from 1:4 to 1:8, from 1:4 to 1:12, from 1:4 to 1:20, from 1:5 to 1:10, from 1:5 to 1:15, from 1:5 to 1:20, from 1:10 to 1:20, or from 1:10 to 1:40. In certain embodiments, multiple genetic loci are targeted hence the ncRNA-sgRNA includes a mixture of ncRNA-sgRNA species and the same ratios and ranges are applicable.

Format of ncRNA and Guide RNA

In certain embodiments, the ncRNA and the guide RNA can be delivered as a single molecule, i.e., with the guide RNA fused to the 5′ and/or 3′ end of the ncRNA. The ncRNA may have guide RNAs located at both ends in some embodiments.

In other embodiments, the guide RNA and ncRNA may be provided and/or delivered as separate components. As shown in Example 4, separation of the guide RNA from the ncRNA can result in increased editing efficiency.

In still other embodiments, a ncRNA-gRNA fusion may be co-delivered with a separate guide RNA.

Modified ncRNAs

In still other embodiments, the ncRNAs disclosed herein may be modified by introducing additional RNA motifs into the ncRNAs, e.g., at the 5′ and 3′ termini of the ncRNAs, or even at positions therein between (e.g., in the msr or msd regions) to improve transcriptional production and/or stability and/or function (e.g., RT-DNA production). Such structures may include, but are not limited to RNA hairpins, RNA step-loops, RNA quadruplexes, cap structures, and poly(A) tails, or ribozyme functions and the like. Also, ncRNAs could also be modified to include one or more nuclear localization sequences.

Additional RNA motifs could also improve RT processivity of the ncRNA or enhance ncRNA activity by enhancing RT binding. Addition of dimerization motifs—such as kissing loops or a GNRA tetraloop/tetraloop receptor pair—at the 5′ and 3′ termini of the ncRNA could also result in effective circularization of the ncRNA, improving stability. Additionally, it is envisioned that addition of these motifs could enable the physical separation of ncRNA components, e.g., separation of the msr and msd regions. Short 5′ extensions or 3′ extensions to the ncRNA that form a small toehold hairpin at either or both ends of the ncRNA could also compete favorably against the annealing of intracomplementary regions along the length of the ncRNA. Finally, kissing loops could also be used to recruit other RNAs or proteins to the genomic site and enable swapping of RT activity from one RNA to the other.

ncRNAs could be further improved via directed evolution, in an analogous fashion to how protein function can be improved. Directed evolution could enhance ncRNA recognition by RT and/or reduce off-site targeting and/or indels and/or improve precise editing efficiency.

The present disclosure contemplates any such ways to further improve the stability and/or functionality of the ncRNAs disclosed here.

In some embodiments, the RNAs (including the guide RNAs and the ncRNAs) used in the compositions of the disclosure have undergone a chemical or biological modification to render them more stable. Exemplary modifications to an RNA include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. The phrase “chemical modifications” as used herein, includes modifications which introduce chemistries which differ from those seen in naturally occurring RNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules).

Other suitable polynucleotide modifications that may be incorporated into the RNAs used in the compositions of the disclosure include, but are not limited to, 4′-thio-modified bases: 4′-thio-adenosine, 4′-thio-guanosine, 4′-thio-cytidine, 4′-thio-uridine, 4′-thio-5-methyl-cytidine, 4′-thio-pseudouridine, and 4′-thio-2-thiouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine, and combinations thereof. The term modification also includes, for example, the incorporation of non-nucleotide linkages or modified nucleotides into the mRNA sequences of the present invention (e.g., modifications to one or both of the 3′ and 5′ ends of an mRNA molecule encoding a functional protein or enzyme). Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3′ UTR or the 5′ UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an RNA molecule (e.g., which form secondary structures).

In some embodiments, RNAs (e.g., ncRNAs) include a 5′ cap structure. A 5′ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5′ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5′5′5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5′)ppp (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G. Naturally occurring cap structures comprise a 7-methyl guanosine that is linked via a triphosphate bridge to the 5′-end of the first transcribed nucleotide, resulting in a dinucleotide cap of m7G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap is added enzymatically. The cap is added in the nucleus and is catalyzed by the enzyme guanylyl transferase. The addition of the cap to the 5′ terminal end of RNA occurs immediately after initiation of transcription. The terminal nucleoside is typically a guanosine, and is in the reverse orientation to all the other nucleotides, i.e., G(5′)ppp(5′)GpNpNp.

Additional cap analogs include, but are not limited to, a chemical structures selected from the group consisting of m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog (e.g., m2,7GpppG), trimethylated cap analog (e.g., m2,2,7GpppG), dimethylated symmetrical cap analogs (e.g., m7Gpppm7G), or anti reverse cap analogs (e.g., ARCA; m7,2′OmeGpppG, m72′dGpppG, m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs with superior translational properties”, RNA, 9: 1108-1122 (2003)).

Typically, the presence of a “tail” serves to protect the RNA (e.g., ncRNA) from exonuclease degradation. A poly A or poly U tail is thought to stabilize natural messengers and synthetic sense RNA. Therefore, in certain embodiments a long poly A or poly U tail can be added to an RNA molecule thus rendering the RNA more stable. Poly A or poly U tails can be added using a variety of art-recognized techniques. For example, long poly A tails can be added to synthetic or in vitro transcribed RNA using poly A polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). A transcription vector can also encode long poly A tails. In addition, poly A tails can be added by transcription directly from PCR products. Poly A may also be ligated to the 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1991 edition)).

Typically, the length of a poly A or poly U tail can be at least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides. In some embodiments, a poly-A tail on the 3′ terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3′ poly(C) tail structure. A suitable poly-C tail on the 3′ terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). The poly-C tail may be added to the poly-A or poly U tail or may substitute the poly-A or poly U tail.

RNAs according to the present disclosure (e.g., ncRNAs) may be synthesized according to any of a variety of known methods. For example, RNAs according to the present invention may be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor. The exact conditions will vary according to the specific application. An improved method of IVT of a ncRNA is disclosed in Example 5 herein.

In a particular embodiment (as exemplified in Example 6 herein), the ncRNAs can comprise an MS2 modification, as specific RNA hairpin structure recognized in nature by a certain MS2-binding protein. This domain can help to stabilize the ncRNA and improve the editing efficiency. The disclosure contemplates other similar modifications. A review of other such MS2-like domains are described in the art, for example, in Johansson et al., “RNA recognition by the MS2 phage coat protein,” Sem Virol., 1997, Vol. 8(3): 176-185; Delebecque et al., “Organization of intracellular reactions with rationally designed RNA assemblies,” Science, 2011, Vol. 333: 470-474; Mali et al., “Cas9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol., 2013, Vol. 31: 833-838; and Zalatan et al., “Engineering complex synthetic transcriptional programs with CRISPR RNA scaffolds,” Cell, 2015, Vol. 160: 339-350, each of which are incorporated herein by reference in their entireties. Other systems include the PP7 hairpin, which specifically recruits the PCP protein, and the “com” hairpin, which specifically recruits the Com protein. See Zalatan et al. The nucleotide sequence of the MS2 hairpin (or equivalently referred to as the “MS2 aptamer”) is:

GCCAACATGAGGATCACCCATGTCTGCAGGGCC

Lipid Nanoparticles

In some embodiments, the lipid delivery sytem includes lipid nanoparticles (LNP). In some embodiments the LNP are small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the non-LNP environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. LNP membranes may be lamellar or non-lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, LNPs may comprise a nucleic acid (e.g. engineered retron) into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.

In some embodiments, an LNP of the present disclosure comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a phospholipid. In alternative embodiments, an LNP comprises an ionizable lipid, a structural lipid, a PEGylated lipid (aka PEG lipid), and a zwitterionic amino acid lipid. In some embodiments, an LNP further comprises a 5^(th) lipid, besides any of the aforementioned lipid components. In some embodiments, the LNP encapsulates one or more elements of the active agent of the present disclosure. In some embodiments, an LNP further comprises a targeting moiety covalently or non-covalently bound to the outer surface of the LNP. In some embodiments, the targeting moiety is a targeting moiety that binds to, or otherwise facilitates uptake by, cells of a particular organ system.

In some embodiments, an LNP has a diameter of at least about 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, or 90 nm. In some embodiments, an LNP has a diameter of less than about 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, or 160 nm. In some embodiments, an LNP has a diameter of less than about 100 nm. In some embodiments, an LNP has a diameter of less than about 90 nm. In some embodiments, an LNP has a diameter of less than about 80 nm. In some embodiments, an LNP has a diameter of about 60-100 nm. In some embodiments, an LNP has a diameter of about 75-80 nm.

In some embodiments, the lipid nanoparticle compositions of the present disclosure are described according to the respective molar ratios of the component lipids in the formulation. As a non-limiting example, the mol-% of the ionizable lipid may be from about 10 mol-% to about 80 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 30 mol-% to about 60 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 35 mol-% to about 55 mol-%. As a non-limiting example, the mol-% of the ionizable lipid may be from about 40 mol-% to about 50 mol-%.

In some embodiments, the mol-% of the phospholipid may be from about 1 mol-% to about 50 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 2 mol-% to about 45 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 3 mol-% to about 40 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 4 mol-% to about 35 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 30 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 10 mol-% to about 20 mol-%. In some embodiments, the mol-% of the phospholipid may be from about 5 mol-% to about 20 mol-%.

In some embodiments, the mol-% of the structural lipid may be from about 10 mol-% to about 80 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 20 mol-% to about 70 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 30 mol-% to about 60 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 35 mol-% to about 55 mol-%. In some embodiments, the mol-% of the structural lipid may be from about 40 mol-% to about 50 mol-%.

In some embodiments, the mol-% of the PEG lipid may be from about 0.1 mol-% to about 10 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.2 mol-% to about 5 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 3 mol-%. In some embodiments, the mol-% of the PEG lipid may be from about 1 mol-% to about 2 mol-%. In some embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%.

i. Ionizable Lipids

In some embodiments, an LNP disclosed herein comprises an ionizable lipid. In some embodiments, an LNP comprises two or more ionizable lipids.

In some embodiments, an ionizable lipid has a dimethylamine or an ethanolamine head. In some embodiments, an ionizable lipid has an alkyl tail. In some embodiments, a tail has one or more ester linkages, which may enhance biodegradability. In some embodiments, a tail is branched, such as with 3 or more branches. In some embodiments, a branched tail may enhance endosomal escape. In some embodiments, an ionizable lipid has a pKa between 6 and 7, which may be measured, for example, by TNS assay.

In some embodiments, an ionizable lipid has a structure of any of the formulas disclosed below, and all formulas disclosed in a reference publication and patent application publication cited below. In some embodiments, an ionizable lipid comprises a head group of any structure or formula disclosed below. In some embodiments, an ionizable lipid comprises a bridging moiety of any structure or formula disclosed below. In some embodiments, an ionizable lipid comprises any tail group, or combination of tail groups disclosed below. The present disclosure contemplates all permutations and combinations of head group, bridging moiety and tail group, or tail groups, disclosed herein.

In some embodiments, a head, tail, or structure of an ionizable lipid is described in US patent application US20170210697A1.

In some embodiments, a compound has a structure according to formula 1:

wherein: R¹ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, YR″, and —R″M′R′; R² and R³ are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, —R*YR″, —YR″, and —R*OR″, or R² and R³, together with the atom to which they are attached, form a heterocycle or carbocycle; R⁴ is selected from the group consisting of a C3.6 carbocycle, —(CH₂)nQ, —(CH2)nCHQR, —CHQR, CO(R)₂, and unsubstituted C1.6 alkyl, where Q is selected from a carbocycle, heterocycle, —OR, —O(CH₂)nN(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —N(R)R⁸, —O(CH₂)_(n)OR, —N(R)C(═NR⁹)N(R)₂—N(R)C(═CHR⁹)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(—NR)N(R)—N(OR)C(═CHR)N(R)₂, —C(═NR⁹)N(R)₂, —C(═NR⁹)R, —C(O)N(R)OR, and —C(R)N(R)₂, C(O)OR, and each n is independently selected from 1, 2, 3, 4, and 5 or a head group disclosed in Table 1; each R⁵ is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R⁶ is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)—, —S—S—, an aryl group, and a heteroaryl group; R⁷ is selected from the group consisting of C1-3alkyl, C2-3 alkenyl, and H; R⁸ is selected from the group consisting of C3-6 carbocycle and heterocycle; R⁹ is selected from the group consisting of H. CN, NO₂, C1-6 alkyl, —OR, —S(O)2R, —S(O)₂N(R)₂, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R′ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, —R*YR″, —YR″, and H; each R″ is independently selected from the group consisting of C3-14 alkyl, C3-14 alkenyl, and H; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl: each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; each Q is —OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R⁸, —NHC(═NR⁹)N(R)₂, —NHC(═CHR)N(R⁹)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13: and wherein when R⁴ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then (i) Q is not —N(R), when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, R⁴ is in Table 1.

In some embodiments, R⁴ in formula 1 is selected from head groups 1-47.

TABLE 1 Ionizable lipid head groups Head number Structure 1

2

3

4

5

6

7

8

9

10

11

12

13

04

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

64

65

66

67

68

In some embodiments, a subset of the compounds of formula 1 are also described by formula 1b:

wherein 1 is selected from 1, 2, 3, 4, and 5; M¹ is a bond or M′; R⁴ is unsubstituted C1-3 alkyl, or —(CH2)nQ, in which n is 2, 3, or 4, and Q is —OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R⁸, —NHC(═NR⁹)N(R)₂, —NHC(═CHR)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and a heteroaryl group; and R² and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. In some embodiments, a head, tail, or structure of an ionizable lipid is described in international patent application PCT/US2018/058555.

In some embodiments, an ionizable lipid has a structure according to formula 2:

wherein: one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O—, and the other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O— or a direct bond; Ra is H or C1-C12 alkyl; R^(1a) and R^(1b) are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^(1a) is H or C1-C12 alkyl, and R^(1b) together with the carbon atom to which it is bound is taken together with an adjacent R^(1b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(2a) and R^(2b) are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^(2a) is H or C1-C12 alkyl, and R^(2b) together with the carbon atom to which it is bound is taken together with an adjacent R^(2b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(3a) and R^(3b) are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^(3a) is H or C1-C12 alkyl, and R^(3b) together with the carbon atom to which it is bound is taken together with an adjacent R^(3b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(4a) and R^(4b) are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^(4a) is H or C1-C12 alkyl, and R^(4b) together with the carbon atom to which it is bound is taken together with an adjacent R^(4b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R⁵ and R⁶ are each independently methyl or cycloalkyl; R⁷ is, at each occurrence, independently H or C1-C12 alkyl; R⁸ and R⁹ are each independently unsubstituted C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; e is 1 or 2; and x is 0, 1 or 2.

In some embodiments, an ionizable lipid has a structure according to formula 3:

wherein: one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O—, and the other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O— or a direct bond; G1 is C1-C2 alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NRaC(═O)— or a direct bond: G2 is —C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NRa— or a direct bond; G3 is C1-C6 alkylene; Ra is H or C1-C12 alkyl; R^(1a) and R^(1b) are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^(1a) is H or C1-C12 alkyl, and R^(1b) together with the carbon atom to which it is bound is taken together with an adjacent R^(1b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(2a) and R^(2b) are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^(2a) is H or C1-C12 alkyl, and R^(2b) together with the carbon atom to which it is bound is taken together with an adjacent R^(2b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(3a) and R^(3b) are, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R^(3a) is H or C1-C12 alkyl, and R^(3b) together with the carbon atom to which it is bound is taken together with an adjacent R^(3b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(4a) and R^(4b) are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R⁴¹ is H or C1-C12 alkyl, and R^(4b) together with the carbon atom to which it is bound is taken together with an adjacent R^(b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R⁵ and R⁶ are each independently H or methyl; R⁷ is C4-C20 alkyl; R⁸ and R⁹ are each independently C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2. In some embodiments, an ionizable lipid has a structure according to formula 4:

wherein: one of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O—, and the other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NRaC(═O)—, —C(═O)NRa—, NRaC(═O)NRa—, —OC(═O)NRa— or —NRaC(═O)O— or a direct bond; G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G³ is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene; Ra is H or C1-C12 alkyl; R¹ and R² are each independently C6-C24 alkyl or C6-C24 alkenyl; R³ is H, OR5, CN, —C(═O)OR4, —OC(═O)R⁴ or —NR5C(═O)R4; R⁴ is C1-C12 alkyl; R⁵ is H or C1-C6 alkyl; and x is 0, 1 or 2.

In some embodiments, an ionizable lipid has a structure according to formula 5:

wherein: one of G or G² is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)y, —S—S—, —C(═O)S—, SC(═O)—, —N(Ra)C(═O)—, —C(═O)N(Ra)—, —N(Ra)C(═O)N(Ra)—, —OC(═O)N(Ra)— or —N(Ra)C(═O)O—, and the other of G1 or G2 is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)y, —S—S—, —C(═O)S—, —SC(═O)—, —N(Ra)C(═O)—, —C(═O)N(Ra)—, —N(Ra)C(═O)N(Ra)—, —OC(═O)N(Ra)— or —N(Ra)C(═O)O— or a direct bond; L is, at each occurrence, ˜O(C═O)—, wherein ˜ represents a covalent bond to X;

X is CRa;

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R¹ and R² have, at each occurrence, the following structure, respectively:

-   a¹ and a² are, at each occurrence, independently an integer from 3     to 12;     b¹ and b² are, at each occurrence, independently 0 or 1;     c¹ and c² are, at each occurrence, independently an integer from 5     to 10;     d¹ and d² are, at each occurrence, independently an integer from 5     to 10;     y is, at each occurrence, independently an integer from 0 to 2; and     n is an integer from 1 to 6,     wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl,     alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy,     alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted     with one or more substituent.

In some embodiments, an ionizable lipid has a structure according to formula 6:

wherein: one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)y, —S—S—, —C(═O)S—, SC(═O)—, —N(Ra)C(═O)—, —C(═O)N(Ra)—, —N(Ra)C(═O)N(Ra)—, —OC(═O)N(Ra)— or —N(Ra)C(═O)O—, and the other of G1 or G2 is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)y-, —S—S—, —C(═O)S—, —SC(═O)—, —N(Ra)C(═O)—, —C(═O)N(Ra)—, —N(Ra)C(═O)N(Ra)—, —OC(═O)N(Ra)— or —N(Ra)C(═O)O— or a direct bond; L is, at each occurrence, ˜O(C═O)—, wherein ˜ represents a covalent bond to X;

X is CRa;

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; Ra is, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R¹ and R² have, at each occurrence, the following structure, respectively:

R′ is, at each occurrence, independently H or C1-C12 alkyl; a¹ and a² are, at each occurrence, independently an integer from 3 to 12; b¹ and b² are, at each occurrence, independently 0 or 1; c¹ and c² are, at each occurrence, independently an integer from 2 to 12; d¹ and d² are, at each occurrence, independently an integer from 2 to 12; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein a¹, a², c¹, c², d¹ and d² are selected such that the sum of a¹+c¹+d¹ is an integer from 18 to 30, and the sum of a²+c²+d² is an integer from 18 to 30, and wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent. In certain embodiments of Formula (V), G¹ and G² are each independently —O(C═O)— or —(C═O)O—.

In some embodiments, an ionizable lipid has a disulfide tail.

In some embodiments, an ionizable lipid includes short peptides of 12-15 mer length as head groups.

In some embodiments, the head of an ionizable lipid comprises the structure of Vitamin A, D, E, or K as described in the published Patent Application WO2019232095A1, which is incorporated by herein by reference in its entirety.

In some embodiments, a lipid is described in international patent applications WO2021077067, or WO2019152557, each of which is incorporated herein by reference in its entirety.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2019/0240354, which is incorporated herein by reference in its entirety.

In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula I:

or salts thereof, wherein: R¹ and R² are either the same or different and are independently hydrogen (H) or an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof; R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either the same or different and are independently an optionally substituted C₁₀-C₂₄ alkyl, C₁₀-C₂₄ alkenyl, C₁₀-C₂₄ alkynyl, or C₁₀-C₂₄ acyl, wherein at least one of R⁴ and R⁵ comprises at least two sites of unsaturation; and n is 0, 1, 2, 3, or 4.

In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula II:

wherein R¹ and R² are either the same or different and are independently an optionally substituted C₁₂-C₂₄ alkyl, C₁₂-C₂₄ alkenyl, C₁₂-C₂₄ alkynyl, or C₁₂-C₂₄ acyl; R³ and R⁴ are either the same or different and are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, or R³ and R⁴ may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R⁵ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; m, n, and p are either the same or different and are independently either 0, 1, or 2, with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are either the same or different and are independently O, S, or NH. In some embodiments, q is 2. In some embodiments, the cationic lipid of Formula II is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane, 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane, 2,2-dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane, 2,2-dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane, 2,2-dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane, 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane, 2,2-dioleoyl-4-dimethylaminomethyl-[1,3]-dioxolane, 2,2-distearoyl-4-dimethylaminomethyl-[1,3]-dioxolane, 2,2-dilinoleyl-4-N-morpholino-[1,3]-dioxolane, 2,2-Dilinoleyl-4-trimethylamino-[1,3]-dioxolane chloride, 2,2-dilinoleyl-4,5-bis(dimethylaminomethyl)-[1,3]-dioxolane, 2,2-dilinoleyl-4-methylpiperzine-[1,3]-dioxolane, or mixtures thereof. In some embodiments, the cationic lipid of Formula II is 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane.

In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula III:

or salts thereof, wherein: R and R² are either the same or different and are independently an optionally substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, or C₂-C₆ alkynyl, or R¹ and R² may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from the group consisting of nitrogen (N), oxygen (O), and mixtures thereof, R³ is either absent or is hydrogen (H) or a C₁-C₆ alkyl to provide a quaternary amine; R⁴ and R⁵ are either absent or present and when present are either the same or different and are independently an optionally substituted C₁-C₁₀ alkyl or C₂-C₁₀ alkenyl; and n is 0, 1, 2, 3, or 4.

In some embodiments, the lipids disclosed in US 2019/0240354 are of Formula C:

X-A-Y—Z¹;  (Formula C)

or salts thereof, wherein:

X is —N(H)R or —NR₂;

A is absent, C₁ to C₆ alkyl, C₂ to C₆ alkenyl, or C₂ to C₆ alkynyl, which C₁ to C₆ alkyl, C₂ to C₆ alkenyl, and C₂ to C₆ alkynyl is optionally substituted with one or more groups independently selected from oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0, 1, or 2, and R^(x) and R^(y) are each independently hydrogen, alkyl, or heterocycle, wherein each alkyl and heterocycle of R^(x) and R^(y) may be further substituted with one or more groups independently selected from oxo, halogen, —OH, —CN, alkyl, —OR^(x′), heterocycle, —NR^(x′)R^(y′), —NR^(x′)C(═O)R^(y′), —NR^(x′)SO₂R^(y′), —C(═O)R^(x′), —C(═O)OR^(x′), —C(═O)NR^(x′)R^(y′), —SO_(n′)R^(x′), and —SO_(n′)NR^(x′)R^(y′), wherein n′ is 0, 1, or 2, and R^(x′) and R^(y′) are each independently hydrogen, alkyl, or heterocycle; Y is selected from the group consisting of absent, —C(═O)—, —O—, —OC(═O)—, —C(═O)O—, —N(R^(b))C(═O)—, —C(═O)N(R^(b))—, —N(R^(b))C(═O)O—, and —OC(═O)N(R^(b)).; Z¹ is a C₁ to C₆ alkyl that is substituted with three or four R^(x) groups, wherein each R^(x) is independently selected from C₆ to C₁₁ alkyl, C₆ to C₁₁ alkenyl, and C₆ to C₁₁ alkynyl, which C₆ to C₁₁ alkyl, C₆ to C₁₁ alkenyl, and C₆ to C₁₁ alkynyl is optionally substituted with one or more groups independently selected from oxo, halogen, heterocycle, —CN, —OR, —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0, 1, or 2, and R^(x) and R^(y) are each independently hydrogen, alkyl, or heterocycle, wherein any alkyl and heterocycle of R^(x) and R^(y) may be further substituted with one or more groups independently selected from oxo, halogen, —OH, —CN, alkyl, —OR^(x′), heterocycle, —NR^(x′)R^(y′), —NR^(x′)C(═O)R^(y′), —NR^(x′)SO₂R^(y′), —C(═O)R^(x′), —C(═O)OR^(x′), —C(═O)NR^(x′)R^(y′), —SO_(n′)R^(x′), and —SO_(n′)NR^(x′)R^(y′), wherein n′ is 0, 1, or 2, and R^(x′) and R^(y′) are each independently hydrogen, alkyl, or heterocycle; each R is independently alkyl, alkenyl, or alkynyl, that is optionally substituted with one or more groups independently selected from oxo, halogen, heterocycle, —CN, —OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x), and —SO_(n)NR^(x)R^(y), wherein n is 0, 1, or 2, and R^(x) and R^(y) are each independently hydrogen, alkyl, or heterocycle, wherein any alkyl and heterocycle of Rand R^(y) may be further substituted with one or more groups independently selected from oxo, halogen, —OH, —CN, alkyl, —OR^(x′), heterocycle, —NR^(x′)R^(y′), —NR^(x′)C(═O)R^(y′), —NR^(x′)SO₂R^(y′), —C(═O)R^(x′), —C(═O)OR^(x′), —C(═O)NR^(x′)R^(y′), —SO_(n′)R^(x′), and —SO_(n∝0)NR^(x′)R^(y′), wherein n′ is 0, 1, or 2, and R^(x′) and R^(y′) are each independently hydrogen, alkyl, or heterocycle; and each Ra is H or Ci to Calkyl.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2010/0130588, which is incorporated herein by reference in its entirety.

In some embodiments, the lipids disclosed in US 2010/0130588 are of Formula I:

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls, R³ and R⁴ are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R³ and R⁴ comprises at least two sites of unsaturation. In some embodiments, R³ and R⁴ are both the same, i.e., R³ and R⁴ are both linoleyl (C¹⁸), etc. In some embodiments, R³ and R⁴ are different, i.e., R³ is tetradectrienyl (C₁₄) and R⁴ is linoleyl (C₁₈).

In some embodiments, the lipid of Formula I is 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

In some embodiments, the lipids disclosed in US 2010/0130588 are of Formula II:

wherein R¹ and R² are independently selected and are H or C₁-C₃ alkyls, R³ and R⁴ are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R³ and R⁴ comprises at least two sites of unsaturation.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2021/0087135, which is incorporated herein by reference in its entirety.

In some embodiments, the lipids disclosed in US 2021/0087135 are of Formula (A):

or its N-oxide, or a salt or isomer thereof, wherein R′^(a) is R′^(branched) or R′^(cyclic); wherein

R′^(branched) is:

R′^(cyclic) is:

wherein:

denotes a point of attachment; wherein R^(aα) is H, and R^(aβ), R^(aγ), and R^(aδ) are each independently selected from the group consisting of H, C₂₋₁₂ alkyl, and C₂₋₁₂ alkenyl, wherein at least one of R^(aβ), R^(aγ), and R^(aδ) is selected from the group consisting of C₂₋₁₂ alkyl and C₂₋₁₂ alkenyl; R² and R³ are each C₁₋₁₄ alkyl; R⁴ is selected from the group consisting of —(CH₂)₂OH, —(CH₂)₃OH, —(CH₂)₄OH, —(CH₂)₅OH and

wherein:

denotes a point of attachment; R¹⁰ is N(R)₂; each R is independently selected from the group consisting of C1.6 alkyl, C₂₋₃ alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R⁵ is independently selected from the group consisting of OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R⁶ is independently selected from the group consisting of OH, C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;

R⁷ is H;

M and M′ are each independently selected from the group consisting of —C(O)O— and —OC(O)—; R′ is a C₁₋₁₂ alkyl or C₂₋₁₂ alkenyl; Y^(a) is a C3.6 carbocycle; R*″^(a) is selected from the group consisting of C₁₋₁₅ alkyl and C₂₋₁₅ alkenyl; 1 is selected from the group consisting of 1, 2, 3, 4, and 5; s is 2 or 3; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2021/0128488, which is incorporated herein by reference in its entirety

In some embodiments, the lipids disclosed in US 2021/0128488 are of structure (I):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: L is —O(C═O)R′, —(C═O)OR¹, —C(═O)R¹, —OR¹, —S(O)_(x)R¹, —S—SR¹, —C(′O)SR′, —SC(═O)R′, —NR^(a)C(═O)R¹, —C(═O)NR^(b)R^(c), —NR^(a)C(═)NR^(b)R^(c), —OC(═O)NR^(b)R^(c) or —NR^(a)C(═O)OR¹; L² is —O(C═O)R², —(C═O)OR², —C(═O)R², —OR², —S(O)_(x)R², —S—SR², —C(═O)SR², —SC(═O)R², —NR^(d)C(═O)R², —C(═O)NR^(e)R^(f), —NR^(d)C(═O)NR^(e)R^(f), —OC(═O)NR^(e)R^(f); —NR^(d)C(═O)OR² or a direct bond to R²; G¹ and G² are each independently C₂-C₁₂ alkylene or C₂-C₁₂ alkenylene; G³ is C₁-C₂₄ alkylene, C₂-C₂₄ alkenylene, C₃-C₈ cycloalkylene or C₃-C₈ cycloalkenylene; R^(a), R^(b), R^(d) and R^(e) are each independently H or C₁-C₁₂ alkyl or C₁-C₁₂ alkenyl; R^(c) and R^(f) are each independently C₁-C₁₂ alkyl or C₂-C₁₂ alkenyl; R¹ and R² are each independently branched C₆-C₂₄ alkyl or branched C₆-C₂₄ alkenyl; R³ is —N(R⁴)R⁵; R⁴ is C₁-C₁₂ alkyl; R⁵ is substituted C₁-C₁₂ alkyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted unless otherwise specified.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2020/0121809, which is incorporated herein by reference in its entirety.

In some embodiments the lipids disclosed in US 2020/0121809 have a structure of Formula II:

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L¹ or L² is —O(C═O)—, —(C═)O—, —C(═O)—, —O—, —S(O)x-, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O—, and the other of L¹ or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O— or a direct bond; G¹ is C₁-C₂ alkylene, —(C═O)—, —O(C═O)—, —SC(═O)—, —NR^(a)C(═O)— or a direct bond; G² is —C(═O)—, —(C═O)O—, —C(═O)S—, —C(═O)NR^(a)— or a direct bond; G³ is C₁-C₆ alkylene; R^(a) is H or C₁-C₁₂ alkyl; R^(1a) and R^(1b) are, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or (b) R^(1a) is H or C₁-C₁₂ alkyl, and R^(1b) together with the carbon atom to which it is bound is taken together with an adjacent R^(1b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(2a) and R^(2b) are, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or (b) R^(2a) is H or C₁-C₁₂ alkyl, and R^(2b) together with the carbon atom to which it is bound is taken together with an adjacent R^(2b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(3a) and R^(3b) are, at each occurrence, independently either (a): H or C₁-C₁₂ alkyl; or (b) R^(3a) is H or C₁-C₁₂ alkyl, and R^(3b) together with the carbon atom to which it is bound is taken together with an adjacent R^(3b) and the carbon atom to which it is bound to form a carbon-carbon double bond; R^(4a) and R^(4b) are, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or (b) R^(4a) is H or C₁-C₁₂ alkyl, and R^(4b) together with the carbon atom to which it is bound is taken together with an adjacent R⁴ and the carbon atom to which it is bound to form a carbon-carbon double bond; R⁵ and R⁶ are each independently H or methyl; R⁷ is C₄-C₂₀ alkyl; R⁸ and R⁹ are each independently C₁-C₁₂ alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

In some embodiments, the lipids disclosed in US 2020/0121809 have a structure of Formula III:

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of L or L² is —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)N^(a)— or —NR^(a)C(═O)O—, and the other of L¹ or L² is —O(C═O)—, —(C═)O—, —C(═O)—, —O—, —S(O)_(x)—, —S—S—, —C(═O)S—, SC(═O)—, —NR^(a)C(═O)—, —C(═O)NR^(a)—, NR^(a)C(═O)NR^(a)—, —OC(═O)NR^(a)— or —NR^(a)C(═O)O— or a direct bond; G¹ and G² are each independently unsubstituted C₁-C₁₂ alkylene or C₁-C₁₂ alkenylene; G³ is C₁-C₂₄ alkylene, C₁-C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene; R^(a) is H or C₁-C₁₂ alkyl; R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl; R³ is H, OR⁵, CN, —C(═O)OR⁴, —OC(═O)R⁴ or —NR⁵C(═O)R⁴; R⁴ is C₁-C₁₂ alkyl; R⁵ is H or C₁-C₆ alkyl; and x is 0, 1 or 2.

In some embodiments, the lipids disclosed in US 2020/0121809 have a structure of Formula (IV):

or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═)O—, —C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, SC(═O)—, —N(Ra)C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a))—, —OC(═O)N(R^(a))— or —N(R^(a))C(═O)O—, and the other of G¹ or G² is, at each occurrence, —O(C═O)—, —(C═O)O—, —C(═O)—, —O—, —S(O)_(y)—, —S—S—, —C(═O)S—, —SC(═O)—, —N(R^(a))C(═O)—, —C(═O)N(R^(a))—, —N(R^(a))C(═O)N(R^(a)—, —OC(═O)N(R^(a))— or —N(R^(a))C(═O)O— or a direct bond; L is, at each occurrence, —O(C═O)—, wherein - represents a covalent bond to X;

X is CR^(a);

Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1; R^(a) is, at each occurrence, independently H, C₁-C₁₂ alkyl, C₁-C₁₂ hydroxylalkyl, C₁-C₁₂ aminoalkyl, C₁-C₁₂ alkylaminylalkyl, C₁-C₁₂ alkoxyalkyl, C₁-C₁₂ alkoxycarbonyl, C₁-C₁₂ alkylcarbonyloxy, C1-C₁₂ alkylcarbonyloxyalkyl or C₁-C₁₂ alkylcarbonyl; R is, at each occurrence, independently either: (a) H or C₁-C₁₂ alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond; R¹ and R² have, at each occurrence, the following structure, respectively:

a¹ and a² are, at each occurrence, independently an integer from 3 to 12; b¹ and b² are, at each occurrence, independently 0 or 1; c¹ and c² are, at each occurrence, independently an integer from 5 to 10; d¹ and d² are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2013/0108685, which is incorporated herein by reference in its entirety.

In some embodiments, the lipids disclosed in US 2013/0108685 are represented by the following formula (I):

wherein: R¹ and R² are, the same or different, each linear or branched alkyl, alkenyl or alkynyl having 12 to 24 carbon atoms, or R¹ and R² are combined together to form dialkylmethylene, dialkenylmethylene, dialkynylmethylene or alkylalkenylmethylene, X¹ and X³ are hydrogen atoms, or are combined together to form a single bond or alkylene, X³ is absent or represents alkyl having 1 to 6 carbon atoms, or alkenyl having 3 to 6 carbon atoms, when X³ is absent, Y is absent, a and b are 0, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, and L¹ and L² are —O—, Y is absent, a and b are, the same or different, 0 to 3, and are not 0 at the same time, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, L¹ and L² are, the same or different, —O—, —CO—O— or —O—CO—, Y is absent, a and b are, the same or different, 0 to 3, L³ is a single bond, R³ is a hydrogen atom, and L¹ and L² are, the same or different, —O—, —CO—O— or —O—CO—, or Y is absent, a and b are, the same or different, 0 to 3, L³ is —CO— or —CO—O—, R³ is pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, wherein at least one of the substituents is amino, monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl, and L¹ and L² are, the same or different, —O—, —CO—O— or —O—CO—, and when X³ is alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms, Y is a pharmaceutically acceptable anion, a and b are, the same or different, 0 to 3, L³ is a single bond, R³ is alkyl having 1 to 6 carbon atoms, alkenyl having 3 to 6 carbon atoms, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or alkyl having 1 to 6 carbon atoms or alkenyl having 3 to 6 carbon atoms substituted with 1 to 3 substituent(s), which is(are), the same or different, amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, L¹ and L² are, the same or different, —O—, —CO—O— or —O—CO—).

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2013/0195920, which is incorporated herein by reference in its entirety.

In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (I), which has a branched alkyl at the alpha position adjacent to the biodegradable group (between the biodegradable group and the tertiary carbon):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl); with respect to R¹ and R², (i) R¹ and R² are each, independently, optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R¹⁰; (ii) R¹ and R², together with the nitrogen atom to which they are attached, form an optionally substituted heterocylic ring; or (iii) one of R¹ and R² is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10 member heterocyclic ring or heteroaryl (e.g., a 6-member ring) with (a) the adjacent nitrogen atom and (b) the (R)_(a) group adjacent to the nitrogen atom; each occurrence of R is, independently, —(CR³R⁴)—; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, R¹⁰, alkylamino, or dialkylamino (In some embodiments, each occurrence of R³ and R⁴ are, independently H or C₁-C₄ alkyl); each occurrence of R¹⁰ is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits, (iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) wherein the compound of formula has at most two R¹⁰ groups (preferably at most one R¹⁰ group); the dashed line to Q is absent or a bond; when the dashed line to Q is absent then Q is absent or is —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—, —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—, —N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—OC(O)—; or when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S, preferably O); each occurrence of R⁵ is, independently, H or alkyl (e.g. C₁-C₄ alkyl); X and Y are each, independently, alkylene or alkenylene (e.g., C₄ to C₂₀ alkylene or C₄ to C₂₀ alkenylene); M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O, —S—S—, C(R⁵)═N—, —N═C(R⁵)—, —C(R)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

wherein R¹¹ is a C₂-C₈ alkyl or alkenyl; each occurrence of R^(z) is, independently, C₁-C₈ alkyl (e.g., methyl, ethyl, isopropyl, n-butyl, n-pentyl, or n-hexyl); a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, or 3; and Z¹ and Z² are each, independently, C₈-C14 alkyl or C₈-C₁₄ alkenyl, wherein the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z².

In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (II), which has a branched alkyl at the alpha position adjacent to the biodegradable group (between the biodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl); with respect to R¹ and R², (i) R¹ and R² are each, independently, optionally substituted alkyl, alkenyl, alkynyl, cycloalkylalkyl, heterocycle, or R¹⁰; (ii) R¹ and R², together with the nitrogen atom to which they are attached, form an optionally substituted heterocylic ring; or (iii) one of R¹ and R² is optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, or heterocycle, and the other forms a 4-10 membered heterocyclic ring or heteroaryl (e.g., a 6-member ring) with (a) the adjacent nitrogen atom and (b) the (R)_(a) group adjacent to the nitrogen atom; each occurrence of R is, independently, —(CR³R⁴)—; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, R¹⁰, alkylamino, or dialkylamino (In some embodiments, each occurrence of R³ and R⁴ are, independently H or C₁-C₄ alkyl); each occurrence of R¹⁰ is independently selected from PEG and polymers based on poly(oxazoline), poly(ethylene oxide), poly(vinyl alcohol), poly(glycerol), poly(N-vinylpyrrolidone), poly[N-(2-hydroxypropyl)methacrylamide] and poly(amino acid)s, wherein (i) the PEG or polymer is linear or branched, (ii) the PEG or polymer is polymerized by n subunits, (iii) n is a number-averaged degree of polymerization between 10 and 200 units, and (iv) wherein the compound of formula has at most two R¹⁰ groups (preferably at most one R¹⁰ group); the dashed line to Q is absent or a bond; when the dashed line to Q is absent then Q is absent or is —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—, —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R)—, —N(R)C(O)N(R)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; or when the dashed line to Q is a bond then (i) b is 0 and (ii) Q and the tertiary carbon adjacent to it (C*) form a substituted or unsubstituted, mono- or bi-cyclic heterocyclic group having from 5 to 10 ring atoms (e.g., the heteroatoms in the heterocyclic group are selected from O and S, preferably O); each occurrence of R⁵ is, independently, H or alkyl; X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) or alkenylene, wherein the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the alpha position to the M or M² group M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O, —S—S—, C(R⁵)═N—, —N═C(R⁵)—, —C(R)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

wherein R¹¹ is a C₂-C₈ alkyl or alkenyl; each occurrence of R^(z) is, independently, C₁-C₈ alkyl (e.g., methyl, ethyl, isopropyl); a is 1, 2, 3, 4, 5 or 6; b is 0, 1, 2, or 3; and Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl, wherein (i) the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z²; and (ii) the terminus of at least one of Z¹ and Z² is separated from the group M¹ or M² by at least 8 carbon atoms.

In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (III), which has a branching point at a position that is 2-6 carbon atoms (i.e., at the beta (p), gamma (γ), delta (δ), epsilon (ε) or zeta position (ζ) adjacent to the biodegradable group (between the biodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², R^(z), a, and b are defined as in formula (I); L¹ and L² are each, independently, C₁-C₅ alkylene or C₂-C₅ alkenylene; X and Y are each, independently, alkylene (e.g., C₄ to C₂₀ alkylene or C₆-C₈ alkylene) or alkenylene (e.g., C₄ to C₂₀ alkenylene); and Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl, wherein the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z². and with the proviso that the terminus of at least one of Z¹ and Z² is separated from the group M¹ or M² by at least 8 carbon atoms.

In some embodiments, the cationic lipid disclosed in US 2013/0195920 is a compound of formula (IV), which has a branching point at a position that is 2-6 carbon atoms (i.e., at beta (β), gamma (γ), delta (δ), epsilon (ε) or zeta position (ζ) adjacent to the biodegradable group (between the biodegradable group and the terminus of the tail, i.e., Z¹ or Z²):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M², R^(z), a, and b are defined as in formula (I); L¹ and L² and are each, independently, C₁-C₅ alkylene or C₂-C₅ alkenylene; X and Y are each, independently, alkylene or alkenylene (e.g., C₁₂-C₂₀ alkylene or C₁₂-C₂₀ alkenylene); and each occurrence of Z is independently C₁-C₄ alkyl (preferably, methyl).

For example, in some embodiments, -L¹-C(Z)₃ is —CH₂C(CH₃)₃. In some embodiments, -L¹-C(Z)₃ is —CH₂CH₂C(CH₃)₃.

In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (V), which has an alkoxy or thioalkoxy (i.e., —S-alkyl) group substitution on at least one tail:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², a, and b are defined as in formula (I); X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) or alkenylene, wherein the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the alpha position to the M¹ or M² group; Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl, wherein (i) the C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl of at least one of Z¹ and Z² is substituted by one or more alkoxy (e.g., a C₁-C₄ alkoxy such as —OCH₃) or thioalkoxy (e.g., a C₁-C₄ thioalkoxy such as —SCH₃) groups, and (ii) the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z².

In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (VIA), which has one or more fluoro substituents on at least one tail at a position that is either alpha to a double bond or alpha to a biodegradable group:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R¹, R², R, a, and b are as defined with respect to formula (I); Q is absent or is —O—, —NH—, —S—, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—, —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—, —N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl); and each of R⁹ and R¹⁰ are independently C₁₂-C₂₄ alkyl (e.g., C₁₂-C₂₀ alkyl), C₁₂-C₂₄ alkenyl (e.g., C₁₂-C₂₀ alkenyl), or C₁₂-C₂₄ alkoxy (e.g., C₁₂-C₂₀ alkoxy) (a) having one or more biodegradable groups and (b) optionally substituted with one or more fluorine atoms at a position which is (i) alpha to a biodegradable group and between the biodegradable group and the tertiary carbon atom marked with an asterisk (*), or (ii) alpha to a carbon-carbon double bond and between the double bond and the terminus of the R⁹ or R¹⁰ group; each biodegradable group independently interrupts the C₁₂-C₂₄ alkyl, alkenyl, or alkoxy group or is substituted at the terminus of the C₁₂-C₂₄ alkyl, alkenyl, or alkoxy group, wherein (i) at least one of R⁹ and R¹⁰ contains a fluoro group; (ii) the compound does not contain the following moiety:

wherein

is an optional bond; and (iii) the terminus of R⁹ and R¹⁰ is separated from the tertiary carbon atom marked with an asterisk (*) by a chain of 8 or more atoms (e.g., 12 or 14 or more atoms).

In some embodiments, the terminus of R⁹ and R¹⁰ is separated from the tertiary carbon atom marked with an asterisk (*) by a chain of 18-22 carbon atoms (e.g., 18-20 carbon atoms).

In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (VIB), which has one or more fluoro substituents on at least one tail at a position that is either alpha to a double bond or alpha to a biodegradable group:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, M¹, M², a, and b are defined as in formula (I); X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) or alkenylene, wherein the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the alpha position to the M¹ or M² group; and Z¹ and Z² are each, independently, C₈-C₁₄ alkyl or C₈-C₁₄ alkenyl, wherein said C₈-C₁₄ alkenyl is optionally substituted by one or more fluorine atoms at a position that is alpha to a double bond, wherein at least one of X, Y, Z¹, and Z² contains a fluorine atom.

In some embodiments, the lipids disclosed in US 2013/0195920 are of formula (VII), which has an acetal group as a biodegradable group in at least one tail:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein R′, R¹, R², R, R³, R⁴, R¹⁰, Q, R⁵, a, and b are defined as in formula (I); X and Y are each, independently, alkylene (e.g., C₆-C₈ alkylene) or alkenylene, wherein the alkylene or alkenylene group is optionally substituted with one or two fluorine atoms at the alpha position to the M¹ or M² group M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O, —S—S—, C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(OXCR³R⁴)C(O)—, or

wherein R¹ is a C₄-C₁₀ alkyl or C₄-C₁₀ alkenyl; with the proviso that at least one of M¹ and M² is

and Z¹ and Z² are each, independently, C₄-C₁₄ alkyl or C₄-C₁₄ alkenyl, wherein the alkenyl group may optionally be substituted with one or two fluorine atoms at the alpha position to a double bond which is between the double bond and the terminus of Z¹ or Z².

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2015/0005363, which is incorporated herein by reference in its entirety.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2014/0308304, which is incorporated herein by reference in its entirety.

In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of formula (I):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein Xaa is a D- or L-amino acid residue having the formula —NR^(N)—CR¹R²—(C═O)—, or a peptide of amino acid residues having the formula —{NR^(N)—CR¹R²—(C═O)}_(n)—, wherein n is 2 to 20; R¹ is independently, for each occurrence, a non-hydrogen, substituted or unsubstituted side chain of an amino acid; R² and RN are independently, for each occurrence, hydrogen, an organic group consisting of carbon, oxygen, nitrogen, sulfur, and hydrogen atoms, or any combination of the foregoing, and having from 1 to 20 carbon atoms, C₍₁₋₅₎alkyl, cycloalkyl, cycloalkylalkyl, C₍₃₋₅₎alkenyl, C₍₃₋₅₎alkynyl, C₍₁₋₅₎alkanoyl, C₍₁₋₅₎alkanoyloxy, C₍₁₋₅₎alkoxy, C₍₁₋₅₎alkoxy-C₍₁₋₅₎alkyl, C₍₁₋₅₎alkoxy-C₍₁₋₅₎alkoxy, C₍₁₋₅₎alkyl-amino-C₍₁₋₅₎alkyl-, C₍₁₋₅₎dialkyl-amino-C₍₁₋₅₎alkyl-, nitro-C₍₁₋₅₎alkyl, cyano-C₍₁₋₅₎alkyl, aryl-C₍₁₋₅₎alkyl, 4-biphenyl-C₍₁₋₅₎alkyl, carboxyl, or hydroxyl; Z is NH, O, S, —CH₂S—, —CH₂S(O)—, or an organic linker consisting of 1-40 atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfur atoms (preferably, Z is NH or O); R^(x) and R^(y) are, independently, (i) a lipophilic tail derived from a lipid (which can be naturally-occurring or synthetic), phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the tail optionally includes a steroid; (ii) an amino acid terminal group selected from hydrogen, hydroxyl, amino, and an organic protecting group; or (iii) a substituted or unsubstituted C₍₃₋₂₂₎alkyl, C₍₆₋₁₂₎cycloalkyl, C₍₆₋₁₂₎cycloalkyl-C₍₃₋₂₂₎alkyl, C₍₃₋₂₂₎alkenyl, C₍₃₋₂₂₎alkynyl, C₍₃₋₂₂₎alkoxy, or C₍₆₋₁₂₎-alkoxy-C₍₃₋₂₂₎alkyl; one of R^(x) and R^(y) is a lipophilic tail as defined above and the other is an amino acid terminal group, or both R^(x) and R^(y) are lipophilic tails; at least one of Rand R^(y) is interrupted by one or more biodegradable groups (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)— or

(wherein R¹¹ is a C₂-C₈ alkyl or alkenyl), in which each occurrence of R⁵ is, independently, H or alkyl; and each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, alkylamino, or dialkylamino; or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C₁-C₄ alkyl)); and R^(x) and R^(y) each, independently, optionally have one or more carbon-carbon double bonds. In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of formula (IA):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein Z and Xaa are as defined with respect to formula (I) (the variables which are used in the definition of Xaa, namely R^(N), R¹ and R², are also as defined in formula (I)); each occurrence of R is, independently, —(CR³R⁴)—; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, alkylamino, or dialkylamino (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C₁-C₄ alkyl); or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group, wherein no more than three R groups in each chain between the —Z-Xaa-C(O)— and Z² moieties are cycloalkyl (e.g., cyclopropyl); Q¹ and Q² are each, independently, absent, —O—, —S—, —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R)C(O)—, —N(R⁵)C(O)N(R⁵)—, or —OC(O)O—; Q³ and Q⁴ are each, independently, H, —(CR³R⁴)—, cycloalkyl, heterocyclyl, heterocyclylalkyl, aryl, heteroaryl, or a cholesterol moiety; each occurrence of A¹, A², A³ and A⁴ is, independently, —(CR⁵R⁵—CR⁵═CR⁵)—; M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

(wherein R¹¹ is a C₂-C₈ alkyl or alkenyl)); each occurrence of R⁵ is, independently, H or alkyl (e.g., C₁-C₄ alkyl); Z² is absent, alkylene or —O—P(O)(OH)—O—; each

attached to Z² is an optional bond, such that when Z² is absent, Q³ and Q⁴ are not directly covalently bound together; c, d, e, f, i, j, m, n, q and r are each, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; g and h are each, independently, 0, 1 or 2; k and l are each, independently, 0 or 1, wherein at least one of k and 1 is 1; o and p are each, independently, 0, 1 or 2; and Q³ and Q⁴ are each, independently, separated from the —Z-Xaa-C(O)— moiety by a chain of 8 or more atoms (e.g., 12 or 14 or more atoms). In some embodiments the lipids disclosed in US 2014/0308304 are of the formula (IC):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein Z and Xaa are as defined with respect to formula (I) (the variables which are used in the definition of Xaa, namely R^(N), R¹ and R², are also as defined in formula (I)); each of R⁹ and R¹⁰ are, independently, alkylene or alkenylene; each of R¹¹ and R¹² are, independently, alkyl or alkenyl, optionally terminated by COOR^(D) wherein each R¹³ is independently unsubstituted alkyl (e.g., C₁-C₄ alkyl such as methyl or ethyl), substituted alkyl (such as benzyl), or cycloalkyl; M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

wherein R¹¹ is a C2-C₈ alkyl or alkenyl, in which each occurrence of R⁵ is, independently, H or alkyl; and each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, alkylamino, or dialkylamino; or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C₁-C₄ alkyl)); R⁹, M¹, and R¹¹ are together at least 8 carbon atoms in length (e.g., 12 or 14 carbon atoms or longer); and R¹⁰, M², and R¹² are together at least 8 carbon atoms in length (e.g., 12 or 14 carbon atoms or longer).

In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of the formula II:

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: s is 1, 2, 3 or 4; and R⁷ is selected from lysyl, ornithyl, 2,3-diaminobutyryl, histidyl and an acyl moiety of the formula:

t is 1, 2 or 3; the NH₃ ⁺ moiety in the acyl moiety in R⁷ is optionally absent; each occurrence of Y⁻ is independently a pharmaceutically acceptable anion (e.g., halide, such as chloride); R⁵ and R⁶ are each, independently a lipophilic tail derived from a naturally-occurring or synthetic lipid, phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside, wherein the tail may contain a steroid; or a substituted or unsubstituted C₍₃₋₂₂₎alkyl, C₍₆₋₁₂₎cycloalkyl, C₍₆₋₁₂₎cycloalkyl-C₍₃₋₂₂₎alkyl, C₍₃₋₂₂₎alkenyl, C₍₃₋₂₂₎alkynyl, C₍₃₋₂₂₎alkoxy, or C₍₆₋₁₂₎alkoxy-C₍₃₋₂₂₎alkyl; at least one of R⁵ and R⁶ is interrupted by one or more biodegradable groups (e.g., —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(O)(NR^(a))—, —N(R^(a))C(O)—, —C(S)(NR^(a))—, —N(R^(a))C(O)—, —N(R^(a))C(O)N(R^(a))—, or —OC(O)O—); each occurrence of R is, independently, H or alkyl; and R⁵ and R⁶ each, independently, optionally contain one or more carbon-carbon double bonds.

In some embodiments, the lipids disclosed in US 2014/0308304 are of the formula (IIA):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R⁷ and s are as defined with respect to formula (II); each occurrence of R is, independently, —(CR³R⁴)—; each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, alkylamino, or dialkylamino (in some embodiments, each occurrence of R³ and R⁴ are, independently H or C₁-C₄ alkyl); or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group, wherein no more than three R groups in each chain attached to the nitrogen N* are cycloalkyl (e.g., cyclopropyl); Q¹ and Q² are each, independently, absent, —O—, —S—, —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, or —OC(O)O—; Q³ and Q⁴ are each, independently, H, —(CR³R⁴)—, aryl, cycloalkyl, heterocyclyl, heterocyclylalkyl, heteroaryl, or a cholesterol moiety; each occurrence of A¹, A², A³ and A⁴ is, independently, —(CR⁵R⁵—CR⁵═CR⁵)—; M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R⁵)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)CR³R⁴)C(O)—, or

wherein R¹¹ is a C₂-C₈ alkyl or alkenyl; each occurrence of R⁵ is, independently, H or alkyl; Z is absent, alkyne or —O—P(O)(OH)—O—; each

attached to Z is an optional bond, such that when Z is absent, Q³ and Q⁴ are not directly covalently bound together; c, d, e, f, i, j, m, n, q and r are each, independently, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; g and h are each, independently, 0, 1 or 2; k and l are each, independently, 0 or 1, where at least one of k and 1 is 1; and o and p are each, independently, 0, 1 or 2.

In some embodiments the lipid disclosed in US 2014/0308304 are of the formula (IIC):

or a salt thereof (e.g., a pharmaceutically acceptable salt thereof), wherein: R⁷ and s are as defined with respect to formula (II); each of R⁹ and R¹⁰ are independently alkyl (e.g., C₁₂-C₂₄ alkyl) or alkenyl (e.g., C₁₂-C₂₄ alkenyl); each of R¹¹ and R¹² are independently alkyl or alkenyl, optionally terminated by COOR^(D) where each R¹³ is independently alkyl (e.g., C₁-C₄ alkyl such as methyl or ethyl); M¹ and M² are each, independently, a biodegradable group (e.g., —OC(O)—, —C(O)O—, —SC(O)—, —C(O)S—, —OC(S)—, —C(S)O—, —S—S—, —C(R⁵)═N—, —N═C(R⁵)—, —C(R⁵)═N—O—, —O—N═C(R⁵)—, —C(O)(NR⁵)—, —N(R⁵)C(O)—, —C(S)(NR⁵)—, —N(R)C(O)—, —N(R⁵)C(O)N(R⁵)—, —OC(O)O—, —OSi(R⁵)₂O—, —C(O)(CR³R⁴)C(O)O—, —OC(O)(CR³R⁴)C(O)—, or

wherein R¹¹ is a C₂-C₈ alkyl or alkenyl; in which each occurrence of R⁵ is, independently, H or alkyl; and each occurrence of R³ and R⁴ are, independently H, halogen, OH, alkyl, alkoxy, —NH₂, alkylamino, or dialkylamino; or R³ and R⁴, together with the carbon atom to which they are directly attached, form a cycloalkyl group (in some embodiments, each occurrence of R³ and R⁴ are, independently, H or C₁-C₄ alkyl)); R⁹, M¹, and R¹¹ are together at least 8 carbons atoms in length (e.g., 12 or 14 carbon atoms or longer); and R¹⁰, M², and R¹² are together at least 8 carbons atoms in length (e.g., 12 or 14 carbon atoms or longer).

In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of the formula (4):

wherein:

X is N or P;

R¹, R², R, a, b, M¹, and M² are as defined with respect to formula (I); Q is absent or is —O—, —NH—, —S—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—, —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—, —N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl); each of R⁹ and R¹⁰ are independently alkylene, or alkenylene; and each of R¹¹ and R¹² are independently alkyl or alkenyl, optionally terminated by COOR¹³ where each R¹³ is independently alkyl (e.g., C₁-C₄ alkyl such as methyl or ethyl); R⁹, M¹, and R¹¹ are together at least 8 carbons atoms in length (e.g., 12 or 14 carbon atoms or longer); and R¹⁰, M², and R¹² are together at least 8 carbons atoms in length (e.g., 12 or 14 carbon atoms or longer).

In some embodiments, the lipid disclosed in US 2014/0308304 is a compound of the formula (5)

wherein:

X is N or P;

R¹, R², R, a, and b are as defined with respect to formula (I); Q is absent or is —O—, —NH—, —S—, —C(O)O—, —OC(O)—, —C(O)N(R⁴)—, —N(R⁵)C(O)—, —S—S—, —OC(O)O—, —O—N═C(R⁵)—, —C(R⁵)═N—O—, —OC(O)N(R⁵)—, —N(R⁵)C(O)N(R⁵)—, —N(R⁵)C(O)O—, —C(O)S—, —C(S)O— or —C(R⁵)═N—O—C(O)—; R′ is absent, hydrogen, or alkyl (e.g., C₁-C₄ alkyl); each of R⁹ and R¹⁰ are independently C₁₂-C₂₄ alkyl or alkenyl substituted at its terminus with a biodegradable group, such as —COOR¹³ where each R¹³ is independently alkyl (preferably C₁-C₄ alkyl such as methyl or ethyl).

In some embodiments the lipids disclosed in US 2014/0308304 are of Formula A:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: n is 0-6 (e.g., n is 0, 1 or 2); R¹ and R² are independently selected from H, (C₁-C₆)alkyl, heterocyclyl, and a polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one or more substituents selected from R′, or R¹ and R² can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 3-7 (e.g., 4-7) members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R′; R³ is selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R′, or R³ can be taken together with R¹ to form a monocyclic heterocycle with 3-7 (e.g., 4-7) members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R′; each occurrence of R⁴, R^(3′) and R^(4′) is independently selected from H, (C₁-C₆)alkyl and O-alkyl, said alkyl is optionally substituted with one or more substituents selected from R′; or R^(3′) and R^(4′) when directly bound to the same carbon atom form an oxo (═O) group, cyclopropyl or cyclobutyl; or R³ and R⁴ form an oxo (═O) group; R⁵ is selected from H and (C₁-C₆)alkyl; or R⁵ can be taken together with R¹ to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R′; each occurrence of R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂; each occurrence of R″ is selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH; L¹ is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl is optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with one or more substituents selected from R′; and L² is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl is optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with one or more substituents selected from R′; with the proviso that the CR^(3′)R^(4′) group when present adjacent to the nitrogen atom in formula A is not a ketone (—C(O)—).

In some embodiments the lipids disclosed in US 2014/0308304 are of formula B:

Formula B

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: n is 0, 1, 2, 3, 4, or 5; R⁶ and R⁷ are each independently (i) C₁-C₄ linear or branched alkyl (e.g., methyl or ethyl) optionally substituted with 1-4 R′, or (ii) C3-C₈ cycloalkyl (e.g., C₃-C₆ cycloalkyl); or R⁶ and R⁷ together with the nitrogen atom adjacent to them form a 3-6 membered ring; L¹ is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; and L² is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; each occurrence of R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂; and each occurrence of R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

In some embodiments, lipids disclosed in US 2014/0308304 are of formula C:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: n is 0, 1, 2, 3, 4, or 5; L¹ is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally has one or more biodegradable groups; each biodegradable group independently interrupts the alkyl or alkenyl group or is substituted at the terminus of the alkyl or alkenyl group, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; and L² is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; each occurrence of R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂; and each occurrence of R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

In some embodiments, the lipid disclosed in US 2014/0308304 are of formula D:

Formula D

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein m is 0, 1, 2, or 3; n is 0, 1, 2, 3, 4, or 5; R⁶ and R⁷ are each independently (i) C₁-C₄ linear or branched alkyl (e.g., methyl or ethyl) optionally substituted with 1-4 R′, or (ii) C₃-C₈ cycloalkyl (e.g., C₃-C₆ cycloalkyl); or R⁶ and R⁷ together with the nitrogen atom adjacent to them form a 3-6 membered ring; L¹ is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; and L² is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups; and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; each occurrence of R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂; and each occurrence of R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

In some embodiments lipid disclosed in US 2014/0308304 are of formula E:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein n is 0, 1, 2, 3, 4, or 5; the group “amino acid” is an amino acid residue; L¹ is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; and L² is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl, said alkyl or alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; each occurrence of R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂; and each occurrence of R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

The amino acid residue in formula E may have the formula —C(O)—C(R⁹)(NH₂), where R⁹ is an amino acid side chain.

In some embodiments, the lipid disclosed in US 2014/0308304 are of formula F:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: R⁶ and R⁷ are independently (i) C₁-C₄ linear or branched alkyl (e.g., methyl or ethyl) optionally substituted with 1-4 R′, or (ii) C₃-C₈ cycloalkyl (e.g., C₃-C₆ cycloalkyl); or R⁶ and R⁷ together with the nitrogen atom adjacent to them form a 3-6 membered ring; L¹ is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; and L² is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; each occurrence of R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂; each occurrence of R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

In some embodiments, the lipid disclosed in US 2014/0308304 are of formula G:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein: n is 0, 1, 2, 3, 4, or 5; q is 1, 2, 3, or 4 R⁶ and R⁷ are independently (i) C₁-C₄ linear or branched alkyl (e.g., methyl or ethyl) optionally substituted with 1-4 R′, or (ii) C₃-C₈ cycloalkyl (e.g., C₃-C₆ cycloalkyl); L¹ is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; and L² is a C₄-C₂₂ alkyl or C₄-C₂₂ alkenyl optionally interrupted by or terminated with one or more biodegradable groups, and said alkyl or alkenyl is optionally substituted with 1-5 substituents selected from R′; each occurrence of R′ is independently selected from halogen, R″, OR″, SR″, CN, CO₂R″ and CON(R″)₂; each occurrence of R″ is independently selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US 2013/0053572, which is incorporated herein by reference in its entirety.

In some embodiments, the lipids disclosed in US 2013/0053572 are of Formula A:

wherein: n is 0, 1 or 2; R¹ and R² are independently selected from H, (C₁-C₆)alkyl, heterocyclyl, and a polyamine, wherein said alkyl, heterocyclyl and polyamine are optionally substituted with one or more substituents selected from R′, or R¹, and R² can be taken together with the nitrogen to which they are attached to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R′; R³ is selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from R′, or R³ can be taken together with R¹ to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R′; R⁴ is selected from H, (C₁-C₆)alkyl and O-alkyl, said alkyl is optionally substituted with one or more substituents selected from R′; R⁵ is selected from H and (C₁-C₆)alkyl; or R⁵ can be taken together with R¹ to form a monocyclic heterocycle with 4-7 members optionally containing, in addition to the nitrogen, one or two additional heteroatoms selected from N, O and S, said monocyclic heterocycle is optionally substituted with one or more substituents selected from R′; R′ is independently selected from halogen, R″, OR″, CN, CO₂R″ and CON(R″)₂; R″ is selected from H and (C₁-C₆)alkyl, wherein said alkyl is optionally substituted with one or more substituents selected from halogen and OH; L₁ is a C₄-C₂₂ alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R′; and L₂ is a C₄-C₂₂ alkenyl, said alkenyl is optionally substituted with one or more substituents selected from R′; or any pharmaceutically acceptable salt or stereoisomer thereof.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in US Application publication US2017/0119904, which is incorporated by reference herein, in its entirety.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in PCT Application publication WO2021/204179, which is incorporated by reference herein, in its entirety.

In some embodiments, an LNP described herein comprises a lipid, e.g., an ionizable lipid, disclosed in PCT Application PCT/US2022/031383, which is incorporated by reference herein, in its entirety.

In some embodiments, an LNP described herein comprises an ionizable lipid of Table 2:

TABLE 2 Exemplary Ionizable Lipids Compound # Structure L-1 

L-2 

L-3 

L-4 

L-5 

L-6 

L-7 

L-8 

L-9 

L-10

ii. Structural Lipids

In some embodiments, an LNP comprises a structural lipid. Structural lipids can be selected from the group consisting of, but are not limited to, cholesterol, fecosterol, fucosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, cholic acid, sitostanol, litocholic acid, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone), or any combinations thereof. In some embodiments, a structural lipid is described in international patent application WO2019152557A1, which is incorporated herein by reference in its entirety.

In some embodiments, a structural lipid is a cholesterol analog. Using a cholesterol analog may enhance endosomal escape as described in Patel et al., Naturally-occuring cholesterol analogues in lipid nanoparticles induce polymorphic shape and enhance intracellular delivery of mRNA, Nature Communications (2020), which is incorporated herein by reference.

In some embodiments, a structural lipid is a phytosterol. Using a phytosterol may enhance endosomal escape as described in Herrera et al., Illuminating endosomal escape of polymorphic lipid nanoparticles that boost mRNA delivery, Biomaterials Science (2020), which is incorporated herein by reference.

In some embodiments, a structural lipid contains plant sterol mimetics for enhanced endosomal release.

iii. PEGylated Lipids

A PEGylated lipid is a lipid modified with polyethylene glycol. In some embodiments, the LNP comprises a compound of Formula I or a pharmaceutically acceptable salt thereof, as described herein above. In some embodiments, the LNP comprises a compound of Formula II or a pharmaceutically acceptable salt thereof, as described herein above.

In some embodiments, an LNP comprises an additional PEGylated lipid or PEG-modified lipid. A PEGylated lipid may be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

In some embodiments, the LNP comprises a PEGylated lipid disclosed in one of US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2015/0203446; US 2017/0210697; US 2014/0200257; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety.

v. Phospholipids

In some embodiments, an LNP of the present disclosure comprises a phospholipid. Phospholipids useful in the compositions and methods may be selected from the non-limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin. In some embodiments, an LNP includes DSPC. In certain embodiments, an LNP includes DOPE. In some embodiments, an LNP includes both DSPC and DOPE.

In some embodiments, a phospholipid tail may be modified in order to promote endosomal escape as described in U.S. 2021/0121411, which is incorporated herein by reference.

In some embodiments, the LNP comprises a phospholipid disclosed in one of US 2019/0240354; US 2010/0130588; US 2021/0087135; WO 2021/204179; US 2021/0128488; US 2020/0121809; US 2017/0119904; US 2013/0108685; US 2013/0195920; US 2015/0005363; US 2014/0308304; US 2013/0053572; WO 2019/232095A1; WO 2021/077067; WO 2019/152557; US 2017/0210697; or WO 2019/089828A1, each of which is incorporated by reference herein in their entirety.

In some embodiments, phospholipids disclosed in US 2020/0121809 have the following structure:

wherein R₁ and R₂ are each independently a branched or straight, saturated or unsaturated carbon chain (e.g., alkyl, alkenyl, alkynyl). vi. Targeting Moieties

In some embodiments, the lipid nanoparticle further comprises a targeting moiety. The targeting moiety may be an antibody or a fragment thereof. The targeting moiety may be capable of binding to a target antigen.

In some embodiments, the pharmaceutical composition comprises a targeting moiety that is operably connected to a lipid nanoparticle. In some embodiments, the targeting moiety is capable of binding to a target antigen. In some embodiments, the target antigen is expressed in a target organ.

In some embodiments, the target antigen is expressed more in the target organ than it is in the liver.

In some embodiments, the targeting moiety is an antibody as described in WO2016189532A1, which is incorporated herein by reference. For example, in some embodiments, the targeted particles are conjugated to a specific anti-CD38 monoclonal antibody (mAb), which allows specific delivery of the siRNAs encapsulated within the particles at a greater percentage to B-cell lymphocytes malignancies (such as MCL) than to other subtypes of leukocytes.

In some embodiments, the lipid nanoparticles may be targeted when conjugated/attached/associated with a targeting moiety such as an antibody.

vii. Zwitterionic Amino Lipids

In some embodiments, an LNP comprises a zwitterionic lipid. In some embodiments, an LNP comprising a zwitterionic lipid does not comprise a phospholipid.

Zwitterionic amino lipids have been shown to be able to self-assemble into LNPs without phospholipids to load, stabilize, and release mRNAs intracellular as described in U.S. Patent Application 20210121411, which is incorporated herein by reference in its entirety. Zwitterionic, ionizable cationic and permanently cationic helper lipids enable tissue-selective mRNA delivery and CRISPR-Cas9 gene editing in spleen, liver and lungs as described in Liu et al., Membrane-destablizing ionizable phospholipids for organ-selective mRNA delivery and CRISPR-Cas gene editing, Nat Mater. (2021), which is incorporated herein by reference in its entirety.

The zwitterionic lipids may have head groups containing a cationic amine and an anionic carboxylate as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013), which is incorporated herein by reference in its entirety. Ionizable lysine-based lipids containing a lysine head group linked to a long-chain dialkylamine through an amide linkage at the lysine α-amine may reduce immunogenicity as described in Walsh et al., Synthesis, Characterization and Evaluation of Ionizable Lysine-Based Lipids for siRNA Delivery, Bioconjug Chem. (2013).

viii. Additional Lipid Components

In some embodiments, the LNP compositions of the present disclosure further comprise one or more additional lipid components capable of influencing the tropism of the LNP. In some embodiments, the LNP further comprises at least one lipid selected from DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200 (see Cheng, et al. Nat Nanotechnol. 2020 April; 15(4): 313-320.; Dillard, et al. PNAS 2021 Vol. 118 No. 52.).

ix. LNP Pharmaceutical Compositions

In some embodiments, a nanoparticle includes an ionizable lipid, a phospholipid, a PEG lipid, and a structural lipid. In certain embodiments, the lipid component of the nanoparticle composition includes about 30 mol % to about 60 mol % ionizable lipid, about 0 mol % to about 30 mol % phospholipid, about 18.5 mol % to about 48.5 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid, provided that the total mol % does not exceed 100%. In some embodiments, the lipid component of the nanoparticle composition includes about 35 mol % to about 55 mol % ionizable lipid, about 5 mol % to about 25 mol % phospholipid, about 30 mol % to about 40 mol % structural lipid, and about 0 mol % to about 10 mol % of PEG lipid. In a particular embodiment, the lipid component includes about 50 mol % ionizable lipid, about 10 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 40 mol % ionizable lipid, about 20 mol % phospholipid, about 38.5 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid. In another particular embodiment, the lipid component includes about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid. In some embodiments, the phospholipid may be DOPE or DSPC. In other embodiments, the PEG lipid may be PEG-DMG and/or the structural lipid may be cholesterol. The amount of active agent in a nanoparticle composition may depend on the size, composition, desired target and/or application, or other properties of the nanoparticle composition as well as on the properties of the active agent. For example, the amount of active agent useful in a nanoparticle composition may depend on the size, sequence, and other characteristics of the active agent. The relative amounts of active agent and other elements (e.g., lipids) in a nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to an enzyme in a nanoparticle composition may be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. The amount of a enzyme in a nanoparticle composition may, for example, be measured using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

In some embodiments, a nanoparticle composition comprising an active agent of the present disclosure is formulated to provide a specific E:P ratio. The E:P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in an RNA active agent. In general, a lower E:P ratio is preferred. The one or more enzymes, lipids, and amounts thereof may be selected to provide an E:P ratio from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the E:P ratio may be from about 2:1 to about 8:1. In other embodiments, the E:P ratio is from about 5:1 to about 8:1. For example, the E:P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1.

The characteristics of a nanoparticle composition may depend on the components thereof. For example, a nanoparticle composition including cholesterol as a structural lipid may have different characteristics than a nanoparticle composition that includes a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For instance, a nanoparticle composition including a higher molar fraction of a phospholipid may have different characteristics than a nanoparticle composition including a lower molar fraction of a phospholipid. Characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition. Nanoparticle compositions may be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) may be used to examine the morphology and size distribution of a nanoparticle composition. Dynamic light scattering or potentiometry (e.g., potentiometric titrations) may be used to measure Zeta potentials. Dynamic light scattering may also be utilized to determine particle sizes. Instruments such as the Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) may also be used to measure multiple characteristics of a nanoparticle composition, Such as particle size, polydispersity index, and Zeta potential.

The mean size of a nanoparticle composition may be between 10s of nm and 100s of nm, e.g., measured by dynamic light scattering (DLS). For example, the mean size may be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the mean size of a nanoparticle composition may be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the mean size of a nanoparticle composition may be from about 70 nm to about 100 nm. In a particular embodiment, the mean size may be about 80 nm. In other embodiments, the mean size may be about 100 nm.

A nanoparticle composition may be relatively homogenous. A polydispersity index may be used to indicate the homogeneity of a nanoparticle composition, e.g., the particle size distribution of the nanoparticle compositions. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. A nanoparticle composition may have a polydispersity index from about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25.

The Zeta potential of a nanoparticle composition may be used to indicate the electrokinetic potential of the composition. For example, the Zeta potential may describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low charges, positive or negative, are generally desirable, as more highly charged species may interact undesirably with cells, tissues, and other elements in the body. In some embodiments, the Zeta potential of a nanoparticle composition may be from about −10 mV to about +20 mV, from about −10 mV to about +15 mV, from about −10 mV to about +10 mV, from about −10 mV to about +5 mV, from about −10 mV to about 0 mV, from about −10 mV to about −5 mV, from about −5 mV to about +20 mV, from about −5 mV to about +15 mV, from about −5 mV to about +10 mV, from about −5 mV to about +5 mV, from about −5 mV to about 0 mV, from about 0 mV, to about +20 mV, from about 0 mV to about +15 mV, from about 0 mV to about +10 mV, from about 0 mV to about +5 mV, from about +5 mV to about +20 mV, from about +5 mV, to about +15 mV, or from about +5 mV to about +10 mV.

The efficiency of encapsulation of a payload describes the amount of payload that is encapsulated or otherwise associated with a nanoparticle composition after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of payload in a solution containing the nanoparticle composition before and after breaking up the nanoparticle composition with one or more organic solvents or detergents. Fluorescence may be used to measure the amount of free payload in a solution. For the nanoparticle compositions described herein, the encapsulation efficiency of a therapeutic and/or prophylactic may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

Lipids and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos. 2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304, 2014/0200257, 2013/086373, 2013/0338210, 2013/0323269, 2013/0245107, 2013/0195920, 2013/0123338, 2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803, 2012/0058188, 2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335, 2011/0060032, 2010/0130588, 2007/0042031, 2006/0240093, 2006/0083780, 2006/0008910, 2005/0175682, 2005/017054, 2005/0118253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2017/117528, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, WO2011/141705, and WO 2001/07548 and Semple et. al, Nature Biotechnology, 2010, 28, 172-176, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

A nanoparticle composition may include any substance useful in pharmaceutical compositions. For example, the nanoparticle composition may include one or more pharmaceutically acceptable excipients or accessory ingredients such as, but not limited to, one or more solvents, dispersion media, diluents, dispersion aids, suspension aids, granulating aids, disintegrants, fillers, glidants, liquid vehicles, binders, surface active agents, isotonic agents, thickening or emulsifying agents, buffering agents, lubricating agents, oils, preservatives, and other species. Excipients such as waxes, butters, coloring agents, coating agents, flavorings, and perfuming agents may also be included. Pharmaceutically acceptable excipients are well known in the art (see for example Remington's The Science and Practice of Pharmacy, 21^(st) Edition, A. R. Gennaro: Lippincott, Williams & Wilkins, Baltimore, Md., 2006). Other different lipids or liposomal formulations including nanoparticles and methods of administration include, but are not limited to, U.S. Patent Publication 20030203865, 20020150626, 20030032615, and 20040048787, which are specifically incorporated by reference to the extent they disclose formulations and other related aspects of administration and delivery of nucleic acids. Methods used for forming particles are also disclosed in U.S. Pat. Nos. 5,844,107, 5,877,302, 6,008,336, 6,077,835, 5,972,901, 6,200,801, and 5,972,900, which are incorporated by reference for those aspects.

In some embodiments, the LNP encapsulates the engineered retron, e.g., an engineered nucleic acid construct, ncRNA, vector system, RNA molecule, and/or engineered nucleic acid-enzyme construct as described herein.

In some embodiments, the lipid nanoparticle comprises: one or more ionizable lipids; one or more structural lipids; one or more PEGylated lipids; and one or more phospholipids. In some embodiments, the one or more ionizable lipids is selected from the group consisting of those disclosed in Table X.

In some embodiments, the one or more structural lipids are selected from the group consisting of cholesterol, fecosterol, beta sitosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, prednisolone, dexamethasone, prednisone, and hydrocortisone. In some embodiments, the one or more PEGylated lipids are selected from the group consisting of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, and PEG-DSPE.

In some embodiments, the one or more phospholipids are selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1.2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoylsn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), and sphingomyelin.

In some embodiments, the lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 40 mol % structural lipid, and about 1.5 mol % of PEG lipid.

In some embodiments, the lipid nanoparticle comprises about 48.5 mol % ionizable lipid, about 10 mol % phospholipid, about 39 mol % structural lipid, and about 2.5 mol % of PEG lipid. In some embodiments, the LNP further comprises a targeting moiety operably connected to the LNP. In some embodiments, the LNP further comprises one or more additional components selected from the group consisting of DDAB, EPC, 14PA, 18BMP, DODAP, DOTAP, and C12-200.

In some embodiments, the engineered retron can be used for gene transfer, which may be performed under ex vivo or in vivo conditions. Ex vivo gene therapy refers to the isolation of cells from a subject, the delivery of a nucleic acid into cells in vitro, and the return of the modified cells back into the subject. This may involve the collection of a biological sample comprising cells from the subject. For example, blood can be obtained by venipuncture, and solid tissue samples can be obtained by surgical techniques according to methods well known in the art.

Usually, but not always, the subject who receives the cells (e.g., the recipient) is also the subject from whom the cells are harvested or obtained, which provides the advantage that the donated cells are autologous. However, cells can be obtained from another subject (e.g., a donor), a culture of cells from a donor, or from established cell culture lines. Accordingly, in some embodiments the cells are allogeneic to the recipient. Cells may be obtained from the same or a different species than the subject to be treated, but preferably are of the same species, and more preferably of the same immunological profile as the subject. Such cells can be obtained, for example, from a biological sample comprising cells from a close relative or matched donor, then transfected with nucleic acids (e.g., comprising an engineered retron), and administered to a subject in need of genome modification, for example, for treatment of a disease or condition.

In other embodiments, the engineered retron can be introduced in vivo (e.g., used in gene therapy) by physically delivering the engineered retron to a subject. Examples of physically introducing the engineered retron includes via injections, electroporation and transfection (e.g., calcium-mediated or liposome tranfection, or the like).

K. Kits

Also provided are kits comprising engineered retrons (e.g., engineered nucleic acid constructs, or engineered nucleic acid-enzyme constructs) as described herein.

In some embodiments, the kit provides an engineered retron construct or a vector system comprising such a retron construct. In some embodiments, the engineered retron construct, included in the kit, comprises a heterologous sequence capable of providing a cell with a nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular recording. Other agents may also be included in the kit such as transfection agents, host cells, suitable media for culturing cells, buffers, and the like.

In the context of a kit, agents can be provided in liquid or solid form in any convenient packaging (e.g., stick pack, dose pack, etc.). The agents of a kit can be present in the same or separate containers. The kits may contain any one or more of the components described herein in one or more containers. The components may be prepared sterilely, packaged in a syringe and shipped refrigerated. Alternatively it may be housed in a vial or other container for storage. A second container may have other components prepared sterilely. Alternatively the kits may include the active agents premixed and shipped in a vial, tube, or other container.

The kits may have a variety of forms, such as a blister pouch, a shrink wrapped pouch, a vacuum sealable pouch, a sealable thermoformed tray, or a similar pouch or tray form, with the accessories loosely packed within the pouch, one or more tubes, containers, a box or a bag. The kits may be sterilized after the accessories are added, thereby allowing the individual accessories in the container to be otherwise unwrapped. The kits can be sterilized using any appropriate sterilization techniques, such as radiation sterilization, heat sterilization, or other sterilization methods known in the art. The kits may also include other components, depending on the specific application, for example, containers, cell media, salts, buffers, reagents, syringes, needles, a fabric, such as gauze, for applying or removing a disinfecting agent, disposable gloves, a support for the agents prior to administration, etc. Some aspects of this disclosure provide kits comprising a nucleic acid construct comprising a nucleotide sequence encoding the various components of the retron-based editing system described herein.

In addition to the above components, the subject kits may further include (in some embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a remote site. In some embodiments, information provided on the website is periodically updated to provide, for example, the most up-to-date information. The written instructions may be in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals or biological products, which can also reflect approval by the agency of manufacture, use or sale for animal administration. As used herein, “promoted” includes all methods of doing business including methods of education, hospital and other clinical instruction, scientific inquiry, drug discovery or development, academic research, pharmaceutical industry activity including pharmaceutical sales, and any advertising or other promotional activity including written, oral and electronic communication of any form, associated with the disclosure.

Other aspects of this disclosure provide kits comprising one or more nucleic acid constructs (e.g., one or more mRNA or circular RNA molecules encoding the components of the retron-based genome editing system) In various embodiments, all nucleic acid constructs can be based on RNA molecules, i.e., and “all-RNA system.” For example, each of the components of the editing system could be expressed from a mRNA molecule, which would be delivered to a target cell by one more more delivery methods (e.g., LNP delivery).

L. Cells

One aspect of the disclosure provides an isolated host cell that includes one or more of the compositions described herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof. In some embodiments, the host cell is a prokaryotic cell, an archaeal cell, or a eukaryotic host cell. In some embodiments, the eukaryotic host cell is a mammalian cell, such as a human cell, a non-human cell, or a non-human mammalian cell. In some embodiments, the host cell is an artificial cell or genetically modified cell. In some embodiments, the host cell is in vitro, such as a tissue culture cell. In some embodiments, the host cell is within a living host organism.

Cells that may contain any of the compositions described herein. The methods described herein are used to deliver recombinant retrons or components thereof into a eukaryotic cell (e.g., a mammalian cell, such as a human cell). In some embodiments, the cell is in vitro (e.g., cultured cell. In some embodiments, the cell is in vivo (e.g., in a subject such as a human subject). In some embodiments, the cell is ex vivo (e.g., isolated from a subject and may be administered back to the same or a different subject).

The present disclosure contemplates the use of any suitable host cell. For example, the cell host can be a mammalian cell. Mammalian cells of the present disclosure include human cells, primate cells (e.g., vero cells), rat cells (e.g., GH3 cells, OC23 cells) or mouse cells (e.g., MC3T3 cells). There are a variety of human cell lines, including, without limitation, human embryonic kidney (HEK) cells, HeLa cells, cancer cells from the National Cancer Institute's 60 cancer cell lines (NCI60), DU145 (prostate cancer) cells, Lncap (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-438 (breast cancer) cells, PC3 (prostate cancer) cells, T47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, SHSY5Y human neuroblastoma cells (cloned from a myeloma) and Saos-2 (bone cancer) cells. In some embodiments, the cells can be human embryonic kidney (HEK) cells (e.g., HEK 293 or HEK 293T cells). In some embodiments, the cells can be stem cells (e.g., human stem cells) such as, for example, pluripotent stem cells (e.g., human pluripotent stem cells including human induced pluripotent stem cells (hiPSCs)). A stem cell refers to a cell with the ability to divide for indefinite periods in culture and to give rise to specialized cells. A pluripotent stem cell refers to a type of stem cell that is capable of differentiating into all tissues of an organism, but not alone capable of sustaining full organismal development. A human induced pluripotent stem cell refers to a somatic (e.g., mature or adult) cell that has been reprogrammed to an embryonic stem cell-like state by being forced to express genes and factors important for maintaining the defining properties of embryonic stem cells (see, e.g., Takahashi and Yamanaka, Cell 126 (4): 663-76, 2006, incorporated by reference herein). Human induced pluripotent stem cells express stem cell markers and are capable of generating cells characteristic of all three germ layers (ectoderm, endoderm, mesoderm).

Some aspects of this disclosure provide cells comprising any of the compositions disclosed herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof. In some embodiments, a host cell is transiently or non-transiently transfected with one or more delivery systems described herein, including virus-based systems, virus-like particle systems, and non-virus-base delivery, including LNPs and liposomes. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject, i.e., ex vivo transfection. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, CIR, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A 172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293. BxPC3. C3H-IOTI/2, C6/36, Ca1-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr −/−, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK 11, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN/OPCT cell lines, Peer, PNT-1A/PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof.

Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, a cell transfected with one or more retron delivery systems described herein is used to establish a new cell line comprising one or more nucleic acid molecules encoding the recombinant retron-based gene editing systems described herein, or encoding at last a component of said systems (e.g., a recombinant ncRNA or a recombinant retron RT).

M. Pharmaceutical Compositions

The engineered retron-based genome editing systems described herein, or one or more components thereof (e.g., engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, guide RNAs, programmable nucleases) may be provided as pharmaceutical compositions. For example, one or more LNPs or other non-virus-based delivery system comprising one or more circular or linear RNA molecules encoding each of the components of the retron-based genome editing system may be formulated as a pharmaceutical composition for administering to a subject in need (e.g., a human in need of gene editing).

Formulations can include, without limitation, saline, liposomes, lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.

Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients. As used herein, the phrase “active ingredient” generally refers an engineered retron as described herein.

A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

Other aspects of the present disclosure relate to pharmaceutical compositions comprising any of the various components of the recombinant retron-based genome editing systems described herein, including, but not limited to, engineered retrons and/or retron components, engineered ncRNAs, engineered msDNA, engineered RT, nucleic acid molecules encoding the engineered retrons and/or retron components, programmable nucleases (e.g., RNA-guided nucleases), guide RNAs, and vector or vector systems encoding the engineered retrons and/or retron components, and any combinations thereof. The term“pharmaceutical composition”, as used herein, refers to a composition formulated for pharmaceutical use. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises additional agents (e.g. for specific delivery, increasing half-life, or other therapeutic compounds).

As used here, the term “pharmaceutically-acceptable carrier” means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the compound from one site (e.g., the delivery site) of the body, to another site (e.g., organ, tissue or portion of the body). A pharmaceutically acceptable carrier is“acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the tissue of the subject (e.g., physiologically compatible, sterile, physiologic pH, etc.).

Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as“excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein.

In some embodiments, the pharmaceutical composition is formulated for delivery to a subject, e.g., for gene editing. Suitable routes of administrating the pharmaceutical composition described herein include, without limitation: topical, subcutaneous, transdermal, intradermal, intralesional, intraarticular, intraperitoneal, intravesical, transmucosal, gingival, intradental, intracochlear, transtympanic, intraorgan, epidural, intrathecal, intramuscular, intravenous, intravascular, intraosseus, periocular, intratumoral, intracerebral, and intracerebroventricular administration.

In some embodiments, the pharmaceutical composition described herein is administered locally to a diseased site (e.g., tumor site). In some embodiments, the pharmaceutical composition described herein is administered to a subject by injection, by means of a catheter, by means of a suppository, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including a membrane, such as a sialastic membrane, or a fiber.

In other embodiments, the pharmaceutical composition described herein is delivered in a controlled release system. In one embodiment, a pump may be used (see, e.g., Langer, 1990, Science 249:1527-1533; Sefton, 1989, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used. (See, e.g., Medical Applications of Controlled Release (Langer and Wise eds., CRC Press, Boca Raton, Fla., 1974); Controlled Drug Bioavailability, Drug Product Design and Performance (Smolen and Ball eds., Wiley, New York, 1984); Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61. See also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). Other controlled release systems are discussed, for example, in Langer, supra.

In some embodiments, the pharmaceutical composition is formulated in accordance with routine procedures as a composition adapted for intravenous or subcutaneous administration to a subject, e.g., a human. In some embodiments, pharmaceutical composition for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the pharmaceutical can also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the pharmaceutical is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the pharmaceutical composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

A pharmaceutical composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the pharmaceutical composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated.

The pharmaceutical composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal or LNP, which is also suitable for parenteral administration. The particles can be of any suitable structure, such as unilamellar or plurilamellar, so long as compositions are contained therein. Compounds can be entrapped in “stabilized plasmid-lipid particles” (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol %) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating (Zhang Y. P. et al., Gene Ther. 1999, 6:1438-47). Positively charged lipids such as N-[1-(2,3-dioleoyloxi)propyl]-N,N,N-trimethyl-amoniummethylsulfate, or “DOTAP,” are particularly preferred for such particles and vesicles. The preparation of such lipid particles is well known. See, e.g., U.S. Pat. Nos. 4,880,635; 4,906,477; 4,911,928; 4,917,951; 4,920,016; and 4,921,757; each of which is incorporated herein by reference.

Further, the pharmaceutical composition can be provided as a pharmaceutical kit comprising (a) a container containing a recombinant retron-based genome editing system or one or more components thereof in lyophilized form and (b) a second container containing a pharmaceutically acceptable diluent (e.g., sterile water) for injection. The pharmaceutically acceptable diluent can be used for reconstitution or dilution of the lyophilized system of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

In another aspect, an article of manufacture containing materials useful for the treatment of the diseases described above is included. In some embodiments, the article of manufacture comprises a container and a label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition that is effective for treating a disease described herein and may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper pierce-able by a hypodermic injection needle. The active agent in the composition is a compound of the invention. In some embodiments, the label on or associated with the container indicates that the composition is used for treating the disease of choice. The article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

N. Use and Methods of Use

The engineered retron comprising the heterologous nucleic acid sequence can be used in a variety of applications, several non-limiting examples of which are described herein. In general, the engineered retron can be used in any suitable organism. In some embodiments, the organism is a eukaryote.

In some embodiments, the organism is an animal. In some embodiments, the animal is a fish, an amphibian, a reptile, a mammal, or a bird. In some embodiments, the animal is a farm animal or agriculture animal. Non-limiting examples of farm and agriculture animals include horses, goats, sheep, swine, cattle, llamas, alpacas, and birds, e.g., chickens, turkeys, ducks, and geese. In some embodiments, the animal is a non-human primate, e.g., baboons, capuchin monkeys, chimpanzees, lemurs, macaques, marmosets, tamarins, spider monkeys, squirrel monkeys, and vervet monkeys. In some embodiments, the animal is a pet. Non-limiting examples of pets include dogs, cats horses, wolfs, rabbits, ferrets, gerbils, hamsters, chinchillas, fancy rats, guinea pigs, canaries, parakeets, and parrots.

In some embodiments, the organism is a plant. Plants that may be transfected with an engineered retron include monocots and dicots. Particular examples include, but are not limited to, corn (maize), sorghum, wheat, sunflower, potato, cotton, rice, soybean, sugarbeet, sugarcane, tobacco, barley, and oilseed rape, Brassica sp., alfalfa, rye, millet, safflower, peanuts, sweet potato, cassava, coffee, coconut, pineapple, citrus trees, cocoa, tea, banana, avocado, fig, guava, mango, olive, papaya, cashew, macadamia, almond, oats, vegetables, ornamentals, and conifers. Vegetables include, but are not limited to, crucifers, peppers, tomatoes, lettuce, green beans, lima beans, peas, and members of the genus Cucumis such as cucumber, cantaloupe, and musk melon. Ornamentals include, but are not limited to, azalea, hydrangea, hibiscus, roses, tulips, daffodils, petunias, carnation, poinsettia, and chrysanthemum.

In some embodiments, heterologous nucleic acid sequences can be added to the subject engineered retron to provide a cell with a heterologous nucleic acid encoding a protein or regulatory RNA of interest, a cellular barcode, a donor polynucleotide suitable for use in gene editing, e.g., by homology directed repair (HDR) or recombination-mediated genetic engineering (recombineering), or a CRISPR protospacer DNA sequence for use in molecular recording, as discussed further below. Such heterologous sequences may be inserted, for example, into the msr locus or the msd locus such that the heterologous sequence is transcribed by the retron reverse transcriptase as part of the msDNA product.

In some embodiments, the engineered retrons described herein may be used for research tools, such as kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening. The kit may comprise one or more reagents in addition to the engineered retron, such as a buffer, a control reagent, a control vector, a control RNA polynucleotide, a reagent for in vitro production of the polypeptide from DNA, and adaptors for sequencing. A buffer can be, for example, a stabilization buffer, a reconstituting buffer, a diluting buffer, a wash buffer, or a buffer for introducing a polypeptide and/or polynucleotide of the kit into a cell. In some instances, a kit can comprise one or more additional reagents specific for plants. One or more additional reagents for plants can include, for example, soil, nutrients, plants, seeds, spores, Agrobacterium, a T-DNA vector, and a pBINAR vector.

Production of Protein or RNA

In some embodiments, the single-stranded msDNA generated by the engineered retron of the invention can be used to produce a desired product of interest in cells.

In some embodiments, the retron is engineered with a heterologous sequence encoding a polypeptide of interest to allow production of the polypeptide from the retron msDNA generated in a cell. The polypeptide of interest may be any type of protein/peptide including, without limitation, an enzyme, an extracellular matrix protein, a receptor, transporter, ion channel, or other membrane protein, a hormone, a neuropeptide, an antibody, or a cytoskeletal protein, a functional fragment thereof, or a biologically active domain of interest. In some embodiments, the protein is a therapeutic protein, therapeutic antibody for use in treatment of a disease, or a template to fix a mutation or mutated exon in the genome.

Non-limiting examples of polypeptides of interest include: growth hormones, insulin-like growth factors (IGF-1), Fat-1, Phytase, xylanase, beta-glucanase, Lysozyme or lysostaphin, Histone deacetylase such as HDAC6, CD163, etc.

In other embodiments, the retron is engineered with a heterologous sequence encoding an RNA of interest to allow production of the RNA from the retron in a cell. The RNA of interest may be any type of RNA including, without limitation, a RNA interference (RNAi) nucleic acid or regulatory RNA such as, but not limited to, a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a small nuclear RNA (snRNA), a long non-coding RNA (IncRNA), an antisense nucleic acid, and the like.

Gene Editing

In some embodiments, the retron is used for genome editing a desired site. A retron is engineered with a heterologous nucleic acid sequence encoding a donor polynucleotide suitable for use with nuclease genome editing system. The nuclease is designed to specifically target a location proximal to the desired edit (the nuclease should be designed such that it will not cut the target once the edit is properly installed). The nuclease (e.g., CAS or non-CAS) is linked to the retron, either by direct fusion to the RT or by fusion of the msDNA to the gRNA (only applicable for RNA-guided nucleases). A heterologous nucleic acid sequence is inserted into the retron msd. See for example FIG. 3 , which shows a marker representing the edit.

In some embodiments, the heterologous nucleic acid sequence has 10-100 or more bp of homologous nucleic acid sequence to the genome on both sides of the desired edit. The desired edit (insertion, deletion, or mutation) is in between the homologous sequence.

In some embodiments, donor polynucleotides comprise a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell. The donor polynucleotide typically comprises a 5′ homology arm that hybridizes to a 5′ genomic target sequence and a 3′ homology arm that hybridizes to a 3′ genomic target sequence. The homology arms are referred to herein as 5′ and 3′ (i.e., upstream and downstream) homology arms, which relate to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5′ and 3′ homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the “5′ target sequence” and “3′ target sequence,” respectively.

The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit can be integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., having sufficient complementary for hybridization) by the 5′ and 3′ homology arms.

In some embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the “5′ target sequence” and “3′ target sequence”) flank a specific site for cleavage and/or a specific site for introducing the intended edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In some embodiments, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

A homology arm can be of any length, e.g. 10 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5′ and 3′ homology arms are substantially equal in length to one another. However, in some instances the 5′ and 3′ homology arms are not necessarily equal in length to one another. For example, one homology arm may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5′ and 3′ homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

The donor polynucleotide may be used in combination with an RNA-guided nuclease, which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to the sequence of a minor allele to target the nuclease-gRNA complex to the site of a mutation. The mutation may comprise an insertion, a deletion, or a substitution. For example, the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation associated with a phenotype or disease of interest. The targeted minor allele may be a common genetic variant or a rare genetic variant. In some embodiments, the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotide polymorphism (SNP). In particular, the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to remove the mutation from a gene. Alternatively, the gRNA can be designed with a sequence complementary to the sequence of a major or wild-type allele to target the nuclease-gRNA complex to the allele for the purpose of genome editing to introduces a mutation into a gene in the genomic DNA of the cell, such as an insertion, deletion, or substitution. Such genetically modified cells can be used, for example, to alter phenotype, confer new properties, or produce disease models for drug screening.

In some embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system Class 1, Type I, II, or III Cas nucleases; Class 2, Type II nuclease (such as Cas9); a Class 2, Type V nuclease (such as Cpf1), or a Class 2, Type VI nuclease (such as C2c2). Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas1O, Cas1Od, CasF, CasG, CasH, Csy1, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx7, Csx14, Csx1O, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.

In some embodiments, a Class 1, type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP 002344900), Campylobacter coli (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC 021284); Prevotella intermedia (NC 017861); Spiroplasma taiwanense (NC 021846); Streptococcus iniae (NC 021314); Belliella baltica (NC 018010); Psychroflexus torquisl (NC O 18721); Streptococcus thermophilus (YP 820832), Streptococcus mutans (WP 061046374, WP 024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus (WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference in their entireties. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacterid. 198(5): 797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA and may further comprise a protospacer adjacent motif (PAM). In some embodiments, the target site comprises 20-30 base pairs in addition to a 3 or more base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two or more other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In some embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele.

In some embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpf1, or Cas12a) is used. Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf1 for targeting than Cas9. Cpf1 is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpf1 have the sequences 5′-YTN-3′ (where “Y” is a pyrimidine and “N” is any nucleobase) or 5′-TTN-3′, in contrast to the G-rich PAM site recognized by Cas9. Cpf1 cleavage of DNA produces double-stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cpf1, see, e.g., Ledford et al. (2015) Nature. 526 (7571):17-17, Zetsche et al. (2015) Cell. 163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.

C2c1 (Cas12b) is another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2c1, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. See, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

In yet another embodiment, an engineered RNA-guided Fok1 nuclease may be used. RNA-guided Fok1 nucleases comprise fusions of inactive Cas9 (dCas9) and the Fok1 endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on Fok1. For a description of engineered RNA-guided Fold nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

In other embodiments, any other Cas enzymes and variants described in other sections of the application (all incorporated herein) can be used similarly.

In some embodiments, the RNA-guided nuclease is provided in the form of a protein, optionally where the nuclease is complexed with a gRNA to form a ribonucleoprotein (RNP) complex. In some embodiments, the RNA-guided nuclease is provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector). In some embodiments, the RNA-guided nuclease and the gRNA are both provided by vectors, such as the vectors and the vector system described in other parts of the application (all incorporated herein by reference). Both can be expressed by a single vector or separately on different vectors. The vectors encoding the RNA-guided nuclease and gRNA may be included in the vector system comprising the engineered retron msr gene, msd gene and ret gene sequences. In some embodiments, the RNA-guided nuclease is fused to the RT and/or the msDNA.

The RNP complex may be administered to a subject or delivered into a cell by methods known in the art, such as those described in U.S. Pat. No. 11,390,884, which is incorporated by reference herein in its entirety. In some embodiments, the endonuclease/gRNA ribonucleoprotein (RNP) complexes are delivered to cells by electroporation. Direct delivery of the RNP complex to a subject or cell eliminates the need for expression from nucleic acids (e.g., transfection of plasmids encoding Cas9 and gRNA). It also eliminates unwanted integration of DNA segments derived from nucleic acid delivery (e.g., transfection of plasmids encoding Cas9 and gRNA). An endonuclease/gRNA ribonucleoprotein (RNP) complex usually is formed prior to administration.

Codon usage may be optimized to further improve production of an RNA-guided nuclease and/or reverse transcriptase (RT) in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease or reverse transcriptase can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guided nuclease or reverse transcriptase is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell.

In some embodiments, the engineered retron used for genome editing with nuclease genome editing systems can further include accessory or enhancer proteins for recombination. Examples of recombination enhancers can include nonhomologous end joining (NHEJ) inhibitors (e.g., inhibitor of DNA ligase IV, a KU inhibitor (e.g., KU70 or KU80), a DNA-PKc inhibitor, or an artemis inhibitor) and homologous directed repair (HDR) promoters, or both, that can enhance or improve more precise genome editing and/or the efficiency of homologous recombination. In some embodiments, the recombination accessory or enhancers can comprise C-terminal binding protein interacting protein (CtIP), cyclinB2, Rad family members (e.g. Rad50, Rad51, Rad52, etc).

CtIP is a transcription factor containing C2H2 zinc fingers that are involved in early steps of homologous recombination. Mammalian CtIP and its orthologs in other eukaryotes promote the resection of DNA double-strand breaks and are essential for meiotic recombination. HDR may be enhanced by using Cas9 nuclease associated (e.g. fused) to an N-terminal domain of CtIP, an approach that forces CtIP to the cleavage site and increases transgene integration by HDR. In some embodiments, an N-terminal fragment of CtIP, called HE for HDR enhancer, may be sufficient for HDR stimulation and requires the CtIP multimerization domain and CDK phosphorylation sites to be active. HDR stimulation by the Cas9-HE fusion depends on the guide RNA used, and therefore the guide RNA will be designed accordingly.

Using the gene editing system described herein, any target gene or sequence in a host cell can be edited or modified for a desired trait, including but not limited to: Myostatin (e.g., GDF8) to increase muscle growth; Pc POLLED to induce hairlessness; KISS1R to induce bore taint; Dead end protein (dnd) to induce sterility; Nano2 and DDX to induce sterility; CD163 to induce PRRSV resistance; RELA to induce ASFV resilience; CD18 to induce Mannheimia (Pasteurella) haemolytica resilience; NRAMP1 to induce tuberculosis resilience; Negative regulators of muscle mass (e.g., Myostatin) to increase muscle mass.

Recombineering

Recombineering (recombination-mediated genetic engineering) can be used in modifying chromosomal as well as episomal replicons in cells, for example, to create gene replacements, gene knockouts, deletions, insertions, inversions, or point mutations. Recombineering can also be used to modify a plasmid or bacterial artificial chromosome (BAC), for example, to clone a gene or insert markers or tags.

The engineered retrons described herein can be used in recombineering applications to provide linear single-stranded or double-stranded DNA for recombination. Homologous recombination may be mediated by bacteriophage proteins such as RecE/RecT from Rac prophage or Redobd from bacteriophage lambda. The linear DNA should have sufficient homology at the 5′ and 3′ ends to a target DNA molecule present in a cell (e.g., plasmid, BAC, or chromosome) to allow recombination.

The linear double-stranded or single-stranded DNA molecule used in recombineering (i.e. donor polynucleotide) comprises a sequence having the intended edit to be inserted flanked by two homology arms that target the linear DNA molecule to a target site for homologous recombination. Homology arms for recombineering typically range in length from 13-300 nucleotides, or 20 to 200 nucleotides, including any length within this range such as 13, 14, 15, 16, 17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 nucleotides in length. In some embodiments, a homology arm is at least 15, at least 20, at least 30, at least 40, or at least 50 or more nucleotides in length. Homology arms ranging from 40-50 nucleotides in length generally have sufficient targeting efficiency for recombination; however, longer homology arms ranging from 150 to 200 bases or more may further improve targeting efficiency. In some embodiments, the 5′ homology arm and the 3′ homology arm differ in length. For example, the linear DNA may have about 50 bases at the 5′ end and about 20 bases at the 3′ end with homology to the region to be targeted.

The bacteriophage homologous recombination proteins can be provided to a cell as proteins or by one or more vectors encoding the recombination proteins, such as the vector or vector system. In some embodiments, one or more vectors encoding the bacteriophage recombination proteins are included in the vector system comprising the engineered retron msr gene, msd gene, and/or ret gene sequences. Additionally, a number of bacterial strains containing prophage recombination systems are available for recombineering, including, without limitation, DY380, containing a defective 1 prophage with recombination proteins exo, bet, and gam; EL250, derived from DY380, which in addition to the recombination genes found in DY380, also contains a tightly controlled arabinose-inducible flpe gene (flpe mediates recombination between two identicalfrt sites); EL350, also derived from DY380, which in addition to the recombination genes found in DY380, also contains a tightly controlled arabinose-inducible ere gene (ere mediates recombination between two identical loxP sites; SW102, derived from DY380, which is designed for BAC recombineering using a galK positive/negative selection; SW105, derived from EL250, which can also be used for galK positive/negative selection, but like EL250, contain an ara-inducible Flpe gene; and SW106, derived from EL350, which can be used for galK positive/negative selection, but like EL350, contains an ara-inducible Cre gene. Recombineering can be carried out by transfecting bacterial cells of such strains with an engineered retron comprising a heterologous sequence encoding a linear DNA suitable for recombineering. For a discussion of recombineering systems and protocols, see, e.g., Sharan et al. (2009) Nat Protoc. 4(2): 206-223, Zhang et al. (1998) Nature Genetics 20: 123-128, Muyrers et al. (1999) Nucleic Acids Res. 27: 1555-1557, Yu et al. (2000) Proc. Natl. Acad. Sci U.S.A. 97 (11):5978-5983; herein incorporated by reference.

Molecular Recording

In some embodiments, the heterologous sequence in the engineered retron construct comprises a synthetic CRISPR protospacer DNA sequence to allow molecular recording. The endogenous CRISPR Cas1-Cas2 system is normally utilized by bacteria and archaea to keep track of foreign DNA sequences originating from viral infections by storing short sequences (i.e., protospacers) that confer sequence-specific resistance to invading viral nucleic acids within genome-based arrays. These arrays not only preserve the spacer sequences but also record the order in which the sequences are acquired, generating a temporal record of acquisition events.

This system can be adapted to record arbitrary DNA sequences into a genomic CRISPR array in the form of “synthetic protospacers” that are introduced into cells using engineered retrons. Engineered retrons carrying the protospacer sequences can be used for integration of synthetic CRISPR protospacer sequences at a specific genomic locus by utilizing the CRISPR system Cas1-Cas2 complex. Molecular recording can be used to keep track of certain biological events by producing a stable genetic memory tracking code. See, e.g., Shipman et al. (2016) Science 353(6298): aafl 175 and International Patent Application Publication No. WO/2018/191525; herein incorporated by reference in their entireties.

In some embodiments, the CRISPR-Cas system is harnessed to record specific and arbitrary DNA sequences into a bacterial genome. The DNA sequences can be produced by an engineered retron within the cell. For example, the engineered retron can be used to produce the protospacers within the cell, which are inserted into a CRISPR array within the cell. The cell may be modified to include one or more engineered returns (or vector systems encoding them) that can produce one or more synthetic protospacers in the cell, wherein the synthetic protospacers are added to the CRISPR array. A record of defined sequences, recorded over many days, and in multiple modalities can be generated.

In some embodiments, the engineered retron comprises an msd protospacer nucleic acid region or an msr protospacer nucleic acid region. In the case of a msr protospacer nucleic acid region, the protospacer sequence is first incorporated into the msr RNA, which is reverse transcribed into protospacer DNA. Double stranded protospacer DNA is produced when two complementary protospacer DNA sequences having complementary sequences hybridize, or when a double-stranded structure (such as a hairpin) is formed in a single stranded protospacer DNA (e.g., a single msDNA can form an appropriate hairpin structure to provide the double stranded DNA protospacer).

In some embodiments, a single stranded DNA produced in vivo from a first engineered retron may be hybridized with a complementary single-stranded DNA produced in vivo from the same retron or a second engineered retron or may form a hairpin structure and then used as a protospacer sequence to be inserted into a CRISPR array as a spacer sequence. The engineered retron(s) should provide sufficient levels of the protospacer sequence within a cell for incorporation into the CRISPR array. The use of protospacers generated within the cell extends the in vivo molecular recording system from only capturing information known to a user, to capturing biological or environmental information that may be previously unknown to a user. For example, an msDNA protospacer sequence in an engineered retron construct may be driven by a promoter that is downstream of a sensor pathway for a biological phenomenon or environmental toxin. The capture and storage of the protospacer sequence in the CRISPR array records the event. If multiple msDNA protospacers are driven by different promoters, the activity of those promoters is recorded (along with anything that may be upstream of the promoters) as well as the relative order of promoter activity (based on the relative position of spacer sequences in the CRISPR array). At any point after the recording has taken place, the CRISPR array may be sequenced to determine whether a given biological or environmental event has taken place and the order of multiple events, given by the presence and relative position of msDNA-derived spacers in the CRISPR array.

In some embodiments, the synthetic protospacer further comprises an AAG PAM sequence at its 5′ end. Protospacers including the 5′ AAG PAM are acquired by the CRISPR array with greater efficiency than those that do not include a PAM sequence.

In some embodiments, Cas1 and Cas2 are provided by a vector that expresses the Cas1 and Cas2 at a level sufficient to allow the synthetic protospacer sequences produced by engineered retrons to be acquired by a CRISPR array in a cell. Such a vector system can be used to allow molecular recording in a cell that lacks endogenous Cas proteins.

Therapeutic Applications

Also provided herein are methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject, using the engineered retron of the invention.

Generally, the methods of diagnosing, prognosing, treating, and/or preventing a disease, state, or condition in or of a subject can include modifying a polynucleotide in a subject or cell thereof using a composition, system, or component thereof of the engineered retron as described herein, and/or include detecting a diseased or healthy polynucleotide in a subject or cell thereof using a composition, system, or component thereof of the engineered retron as described herein.

In some embodiments, the method of treatment or prevention can include using a composition, system, or component of the engineered retron to modify a polynucleotide of an infectious organism (e.g. bacterial or virus) within a subject or cell thereof.

In some embodiments, the method of treatment or prevention can include using a composition, system, or component of the engineered retron to modify a polynucleotide of an infectious organism or symbiotic organism within a subject.

In some embodiments, the composition, system, and components of the engineered retron can be used to develop models of diseases, states, or conditions.

In some embodiments, the composition, system, and components of the engineered retron can be used to detect a disease state or correction thereof, such as by a method of treatment or prevention described herein.

In some embodiments, the composition, system, and components of the engineered retron can be used to screen and select cells that can be used, for example, as treatments or preventions described herein.

In some embodiments, the composition, system, and components thereof can be used to develop biologically active agents that can be used to modify one or more biologic functions or activities in a subject or a cell thereof.

In general, the method can include delivering a composition, system, and/or component of the engineered retron to a subject or cell thereof, or to an infectious or symbiotic organism by a suitable delivery technique and/or composition. Once administered, the components can operate as described elsewhere herein to elicit a nucleic acid modification event. In some embodiments, the nucleic acid modification event can occur at the genomic, epigenomic, and/or transcriptomic level. DNA and/or RNA cleavage, gene activation, and/or gene deactivation can occur.

The composition, system, and components of the engineered retron as described elsewhere herein can be used to treat and/or prevent a disease, such as a genetic and/or epigenetic disease, in a subject; to treat and/or prevent genetic infectious diseases in a subject, such as bacterial infections, viral infections, fungal infections, parasite infections, and combinations thereof, to modify the composition or profile of a microbiome in a subject, which can in turn modify the health status of the subject; to modify cells ex vivo, which can then be administered to the subject whereby the modified cells can treat or prevent a disease or symptom thereof, or to treat mitochondrial diseases, where the mitochondrial disease etiology involves a mutation in the mitochondrial DNA.

Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing gene editing by transforming the subject with the polynucleotide encoding one or more components of the composition, system, or complex or any of polynucleotides or vectors described herein of the engineered retron, and administering them to the subject.

Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing transcriptional activation or repression of multiple target gene loci by transforming the subject with the polynucleotides or vectors described herein, wherein said polynucleotide or vector encodes or comprises one or more components of composition, system, complex or component of the engineered retron, and comprising multiple Cas effectors.

Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing gene editing by transforming the subject with the Cas effector(s), and encoding and expressing in vivo the remaining portions of the composition, system, (e.g., RNA, guides), complex or component of the engineered retron. A suitable repair template may also be provided by the engineered retron as described herein elsewhere.

Also provided is a method of treating a subject, e.g., a subject in need thereof, comprising inducing transcriptional activation or repression by transforming the subject with the systems or compositions herein.

Also provided is a method of inducing one or more polynucleotide modifications in a eukaryotic or prokaryotic cell or component thereof (e.g. a mitochondria) of a subject, infectious organism, and/or organism of the microbiome of the subject. The modification can include the introduction, deletion, or substitution of one or more nucleotides at a target sequence of a polynucleotide of one or more cell(s). The modification can occur in vitro, ex vivo, in situ, or in vivo.

In some embodiments, the method of treating or inhibiting a condition or a disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non-human organism can include manipulation of a target sequence within a coding, non-coding or regulatory element of said genomic locus in a target sequence in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by manipulation of the target sequence and wherein the condition or disease is susceptible to treatment or inhibition by manipulation of the target sequence including providing treatment comprising delivering a composition comprising the particle delivery system or the delivery system or the virus particle of any one of the above embodiment or the cell of any one of the above embodiment.

Also provided herein is the use of the particle delivery system or the delivery system or the virus vector (in viral particle) of any one of the above embodiment or the cell of any one of the above embodiment in ex vivo or in vivo gene or genome editing; or for use in in vitro, ex vivo or in vivo gene therapy.

Also provided herein are particle delivery systems, non-viral delivery systems, and/or the virus particle of any one of the above embodiments or the cell of any one of the above embodiments used in the manufacture of a medicament for in vitro, ex vivo or in vivo gene or genome editing or for use in in vitro, ex vivo or in vivo gene therapy or for use in a method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus associated with a disease or in a method of treating or inhibiting a condition or disease caused by one or more mutations in a genomic locus in a eukaryotic organism or a non-human organism.

In some embodiments, target polynucleotide modification using the subject engineered retron and the associated composition, vectors, system and methods comprises addition, deletion, or substitution of 1-about 10 k nucleotides at each target sequence of said polynucleotide of said cell(s). The modification can include the addition, deletion, or substitution of at least 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 100, 200, 250, 300, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10,000 or more nucleotides at each target sequence.

In some embodiments, formation of system or complex results in cleavage, nicking, and/or another modification of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.

In some embodiments, a method of modifying a target polynucleotide in a cell to treat or prevent a disease can include allowing a composition, system, or component of the subject engineered retron to bind to the target polynucleotide, e.g., to effect cleavage, nicking, or other modification as the composition, system, is capable of said target polynucleotide, thereby modifying the target polynucleotide, wherein the composition, system, or component thereof, complex with a guide sequence, and hybridize said guide sequence to a target sequence within the target polynucleotide, wherein said guide sequence is optionally linked to a tracr mate sequence, which in turn can hybridize to a tracr sequence. In some embodiments, modification can include cleaving or nicking one or two strands at the location of the target sequence by one or more components of the composition, system, or component thereof.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the circulatory system. In some embodiments, the treatment can be carried out by using an AAV or a lentiviral vector to deliver the engineered retron, composition, system, and/or vector described herein to modify hematopoietic stem cells (HSCs) or iPSCs in vivo or ex vivo. In some embodiments, the treatment can be carried out by correcting HSCs or iPSCs as to the disease using a composition, system, herein or a component thereof, wherein the composition, system, optionally includes a suitable HDR repair template (e.g., a template in the msDNA of the engineered retron).

In some embodiments, the treatment or prevention for treating a circulatory system or blood disease can include modifying a human cord blood cell. In some embodiments, the treatment or prevention for treating a circulatory system or blood disease can include modifying a granulocyte colony-stimulating factor-mobilized peripheral blood cell (mPB) with any modification described herein. In some embodiments, the human cord blood cell or mPB can be CD34*. In some embodiments, the cord blood cells or mPB cells modified are autologous. In some embodiments, the cord blood cells or mPB cells are allogenic. In addition to the modification of the disease genes, allogenic cells can be further modified using the composition, system, described herein to reduce the immunogenicity of the cells when delivered to the recipient. The modified cord blood cells or mPB cells can be optionally expanded in vitro. The modified cord blood cell(s) or mPB cells can be derived to a subject in need thereof using any suitable delivery technique.

The composition and system may be engineered to target genetic locus or loci in HSCs. In some embodiments, the components of the systems can be codon-optimized for a eukaryotic cell and especially a mammalian cell, e.g., a human cell, for instance, HSC, or iPSC and sgRNA targeting a locus or loci in HSC, such as circulatory disease, can be prepared. These may be delivered via particles, such as the lipid nanoparticle delivery system described herein. The particles may be formed by the components of the systems herein being admixed.

In some embodiments, after ex vivo modification the HSCs or iPCS can be expanded prior to administration to the subject. Expansion of HSCs can be via any suitable method such as that described by, Lee, “Improved ex vivo expansion of adult hematopoietic stem cells by overcoming CUL4-mediated degradation of HOXB4.” Blood. 2013 May 16; 121(20):4082-9. doi: 10.1182/blood-2012-09-455204. Epub 2013 Mar. 21.

In some embodiments, the HSCs or iPSCs modified are autologous. In some embodiments, the HSCs or iPSCs are allogenic. In addition to the modification of the disease genes, allogenic cells can be further modified using the composition, system, described herein to reduce the immunogenicity of the cells when delivered to the recipient.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat neurological diseases. In some embodiments, the neurological diseases comprise diseases of the brain and CNS.

Delivery options for the diseases in the brain include encapsulation of the systems in the form of either DNA or RNA into liposomes and conjugating to molecular Trojan horses for trans-blood brain barrier (BBB) delivery. Molecular Trojan horses have been shown to be effective for delivery of B-gal expression vectors into the brain of non-human primates. The same approach can be used to delivery vectors or vector systems of the invention. In other embodiments, an artificial virus can be generated for CNS and/or brain delivery.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat hearing diseases or hearing loss in one or both ears. Deafness is often caused by lost or damaged hair cells that cannot relay signals to auditory neurons. In some embodiments, the composition, system, or modified cells can be delivered to one or both ears for treating or preventing hearing disease or loss by any suitable method or technique known in the art, such as US20120328580 (e.g., auricular administration), by intratympanic injection (e.g., into the middle ear), and/or injections into the outer, middle, and/or inner ear; administration in situ, via a catheter or pump (U.S. 2006/0030837) and Jacobsen (U.S. Pat. No. 7,206,639). Also see US20120328580. Cells resulting from such methods can then be transplanted or implanted into a patient in need of such treatment.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases in non-dividing cells. Exemplary non-dividing cells include muscle cells or neurons. In such cells, homologous recombination (HR) is generally suppressed in the G1 cell-cycle phase, but can be turned back on using art-recognized methods, such as Orthwein et al. (Nature. 2015 Dec. 17; 528(7582): 422-426).

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the eye.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat muscle diseases and cardiovascular diseases.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the liver and kidney.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat epithelial and lung diseases.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat diseases of the skin.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat cancer.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used in adoptive cell therapy.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat infectious diseases.

In some embodiments, the engineered retron and the associated compositions, systems, vectors, uses, and methods of use, can be used to treat mitochondrial diseases.

All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

O. Sequences

The instant Specification discloses and claims recombinant retrons and components thereof (e.g., recombinant ncRNAs and recombinant retron RTs), as well as genome modification systems comprising said recombinant retrons and/or recombinant components thereof. Without limitation, such systems include recombinant retron-based genome editing systems, recombineering systems, and cell-recording systems. The Specification also discloses and claims the various components and aspects making up the systems, as well as their uses and applications, including without limitation: (a) recombinant nucleic acid molecules encoding recombinant retrons and retron-based genome modification systems, (b) vector systems (including viral and non-viral) comprising one or more components of said retron-based genome modification systems, including all-DNA vector systems, all-RNA vector systems, and DNA/RNA vector systems, (c) delivery systems for delivering said vector systems and/or components of the retron-based genome modification systems (e.g., lipid particles, lipid nanoparticles, and other delivery vehicle formats), (d) formulations comprising any of the aforementioned components for delivery to cells and/or tissues, including in vitro, in vivo, and ex vivo delivery, (e) cells modified by the recombinant retron-based genome modification systems and methods described herein, and (f) methods of modifying cells by conducting genome editing, recombineering, or cell recording using the herein disclosed retron-based genome modification systems, (g) methods of making the recombinant retrons, retron-based genome modification systems, vectors, and formulations described herein, and (h) pharmaceutical compositions and kits for modifying cells under in vitro, in vivo, and ex vivo conditions.

The recombinant retrons and retron-based genome modification systems described herein can be made, in various embodiments, by introducing one or more modifications into a known retron, e.g., a retron that is publicly known (Group I retrons) or a novel retron that is found in nature but which has not previously been recognized or described (Group II retrons). Examples of such retron sequences (and in some cases sequences of their components) are disclosed herein and summarized below:

TABLE X Previously known retron reverse transcriptases SEQ ID NO: Retron 19413 name (if -to- NCBI Accession^(a) Species/strain applicable) 21341 fig|670897.3.peg.2382 Escherichia coli 2362-75 19413 WP_000111473.1 Escherichia coli Retron- Eco7 (Ec78) fig|286156.4.peg.5031 Photorhabdus australis fig|171439.3.peg.1995 Photorhabdus luminescens subsp. luminescens fig|1004151.3.peg.110 Photorhabdus khanii NC19 fig|1736225.3.peg.2969 Erwinia sp. Leaf53 fig|1897730.3.peg.2912 Citrobacter sp. CFSAN044567 fig|286156.4.peg.5031 Aeromonas australiensis fig|1460083.3.peg.4429 Serratia liquefaciens FK01 fig|585.10.peg.2369 Proteus vulgaris WP_140315795.1 Vibrio parahaemolyticus Retron- Vpa1 (Vp96) ^(g) fig|670.147.peg.3463 Vibrio parahaemolyticus fig|1516159.4.peg.4737 Vibrio coralliirubri fig|190893.12.peg.246 Vibrio coralliilyticus fig|643674.5.peg.820 Paenalcaligenes hominis fig|1122619.3.peg.2381 Oligella ureolytica DSM 18253 fig|29489.5.peg.3423 Aeromonas enteropelogenes fig|1899355.18.peg.3566 Oceanospirillaceae bacterium fig|49186.3.peg.4362 Marinobacterium stanieri fig|672.375.peg.4377 Vibrio vulnificus fig|584.202.peg.1668 Proteus mirabilis fig|394935.10.peg.4407 Chromobacterium haemolyticum fig|1196083.117.peg.637 Snodgrassella alvi fig|1196083.120.peg.2046 Snodgrassella alvi fig|1196083.114.peg.825 Snodgrassella alvi fig|550.250.peg.2975 Enterobacter cloacae fig|680.27.peg.793 Vibrio campbellii fig|1348393.3.peg.352 Pseudoalteromonas sp. H105 fig|644.85.peg.4392 Aeromonas hydrophila fig|1234128.4.peg.4777 Vibrio parahaemolyticus SNUVpS-1 fig|69219.6.peg.2213 Enterobacter cloacae subsp. dissolvens fig|208224.13.peg.2962 Enterobacter kobei fig|672.332.peg.2758 Vibrio vulnificus fig|1777131.3.peg.2267 Chromobacterium sp. F49 fig|945550.3.peg.1167 Vibrio sinaloensis DSM 21326 fig|648.75.peg.922 Aeromonas caviae fig|1238221.3.peg.2053 Vibrio parahaemolyticus VPTS-2009 fig|56192.3.peg.3860 Photobacterium iliopiscarium fig|1806667.7.peg.3169 Marinomonas gallaica fig|272773.3.peg.1019 Salinivibrio costicola subsp. alcaliphilus WP_073265166.1 Pseudomonas punonensis fig|1946584.3.peg.2789 Halomonas sp. UBA3074 fig|2030880.3.peg.665 SAR86 cluster bacterium fig|80854.14.peg.530 Moritella viscosa fig|1902503.3.peg.1072 Marinomonas sp. QM202 fig|1122212.3.peg.1985 Marinospirillum minutulum DSM 6287 fig|40576.4.peg.4387 Xenorhabdus bovienii fig|287094.3.peg.78 Alteromonas addita fig|1805633.3.peg.1469 Acinetobacter sp. SFA fig|1945927.3.peg.1017 Acinetobacter sp. UBA1497 fig|202956.9.peg.1680 Acinetobacter towneri fig|1811612.3.peg.155 Moraxellaceae bacterium REDSEA-S32 B1 fig|573.14330.peg.438 Klebsiella pneumoniae fig|470.1294.peg.971 Acinetobacter baumannii fig|762966.3.peg.2452 Parasutterella excrementihominis YIT 11859 fig|470.3514.peg.1550 Acinetobacter baumannii fig|470.2538.peg.3022 Acinetobacter baumannii fig|48296.130.peg.276 Acinetobacter pittii fig|663.91.peg.4688 Vibrio alginolyticus fig|296199.3.peg.4813 Vibrio gigantis fig|1367490.3.peg.3583 Aliivibrio fischeri ETJB5C fig|326537.3.peg.3698 Colwellia polaris fig|1175631.4.peg.4191 Pectobacterium wasabiae CFBP 3304 WP_001403504.1 Escherichia coli Retron- Eco4 (Ec83) fig|549.21.peg.1734 Pantoea agglomerans fig|140100.3.peg.2972 Vibrio cholerae fig|693153.4.peg.1176 Vibrio atlanticus fig|1238430.3.peg.1911 Vibrio nigripulchritudo AM115 fig|1123036.3.peg.144 Psychromonas arctica DSM 14288 fig|173990.3.peg.3319 Rheinheimera pacifica fig|1869214.4.peg.3809 Rheinheimera sp. fig|1898113.7.peg.1514 Idiomarinaceae bacterium fig|29484.39.peg.1876 Yersinia frederiksenii fig|1761793.3.peg.274 Marinobacter sp. DSM 26671 fig|587.48.peg.2666 Providencia rettgeri fig|573.4147.peg.1684 Klebsiella pneumoniae fig|1263833.3.peg.2872 Serratia marcescens VGH107 fig|1690502.3.peg.467 Pantoea sp. CFSAN033090 fig|1029989.3.peg.5037 Salmonella enterica subsp. enterica serovar Agona str. 0292 fig|211759.3.peg.770 Serratia marcescens fig|29483.5.peg.2283 Yersinia aldovae fig|1268238.3.peg.3466 Escherichia coli O5:K4(L):H4 str. ATCC 23502 fig|548.121.peg.2368 Klebsiella aerogenes fig|196024.6.peg.1825 Aeromonas dhakensis fig|386429.3.peg.3784 Pseudoalteromonas sp. BSi20495 fig|666.2089.peg.3167 Vibrio cholerae WP_159353404.1 Vibrio cholerae Retron- Vch1 (Vc95) fig|670.362.peg.2186 Vibrio parahaemolyticus fig|615.398.peg.1671 Serratia marcescens fig|571.188.peg.5401 Klebsiella oxytoca fig|1389422.3.peg.2794 Klebsiella pneumoniae LAU-KP1 fig|1082704.3.peg.1242 Lonsdalea britannica fig|1686379.3.peg.3365 Citrobacter sp. MGH104 fig|83655.55.peg.221 Leclercia adecarboxylata fig|550.532.peg.617 Enterobacter cloacae fig|349965.6.peg.153 Yersinia intermedia ATCC 29909 fig|1947028.3.peg.31 Pantoea sp. UBA2708 fig|29484.34.peg.3725 Yersinia frederiksenii fig|314608.4.peg.222 Shewanella benthica KT99 fig|585.16.peg.3620 Proteus vulgaris fig|1117313.3.peg.4128 Pseudoalteromonas arctica A 37-1-2 fig|1236543.3.peg.1328 Shewanella putrefaciens JCM 20190 = NBRC 3908 fig|550.520.peg.1818 Enterobacter cloacae fig|592316.4.peg.43 Pantoea sp. At-9b fig|1903177.3.peg.4556 Vibrio sp. 10N.261.45.E1 fig|1435069.3.peg.925 Vibrio tritonius fig|666.3258.peg.1211 Vibrio cholerae fig|1579504.3.peg.1822 Shewanella sp. ECSMB14102 fig|727.548.peg.1576 Haemophilus influenzae EIJ70524.1 Haemophilus parahaemolyticus HK385 fig|1121935.3.peg.14 Hahella ganghwensis DSM 17046 fig|400668.8.peg.2509 Marinomonas sp. MWYL1 fig|1777491.3.peg.1212 Alteromonas sp. Mac1 fig|2013797.3.peg.1728 Gammaproteobacteria bacterium HGW-Gammaproteobacteria-15 fig|1008297.7.peg.4158 Salmonella enterica subsp. enterica serovar Typhimurium str. 798 EDM6246721.1 Salmonella enterica subsp. Retron- enterica serovar Typhimurium Sen2 (St85) fig|421.19.peg.3278 Methylomonas methanica fig|758.17.peg.102 Rodentibacter pneumotropicus fig|726.60.peg.864 Haemophilus haemolyticus fig|1035188.3.peg.348 Haemophilus pittmaniae HK 85 fig|670.79.peg.3738 Vibrio parahaemolyticus fig|1481663.12.peg.913 Vibrio metoecus fig|1123402.3.peg.611 Thorsellia anophelis DSM 18579 fig|668.83.peg.3088 Aliivibrio fischeri fig|290110.6.peg.2319 Xenorhabdus budapestensis fig|568766.10.peg.822 Dickeya sp. NCPPB 3274 fig|470.4268.peg.2217 Acinetobacter baumannii fig|1977881.3.peg.1569 Acinetobacter sp. ANC 4470 fig|548.171.peg.2395 Klebsiella aerogenes fig|584.105.peg.1823 Proteus mirabilis fig|1275975.3.peg.1756 Salmonella enterica subsp. enterica serovar Newport str. Henan_3 fig|615.474.peg.3994 Serratia marcescens fig|61647.13.peg.3699 Pluralibacter gergoviae fig|549.22.peg.222 Pantoea agglomerans fig|991944.3.peg.3216 Vibrio cholerae HE-25 WP_001022871.1 Vibrio cholerae Retron- Vch2 (Vc81) fig|1638949.3.peg.1051 Vibrio sp. ECSMB14106 fig|73010.3.peg.2815 Aeromonas encheleia fig|1444141.3.peg.3893 Escherichia coli 3-373-03_S3_C1 fig|232.5.peg.l080 Alteromonas sp. fig|1175295.3.peg.21 Pseudoalteromonas sp. PAMC 22718 fig|265726.7.peg.3430 Photobacterium halotolerans WP_009585554.1 Acinetobacter fig|2004649.3.peg.1632 Acinetobacter sp. WCHA29 fig|1324350.3.peg.2817 Acinetobacter equi fig|2048003.3.peg.1682 Alteromonas flava fig|571.171.peg.5963 Klebsiella oxytoca fig|573.4060.peg.3574 Klebsiella pneumoniae fig|1173850.3.peg.2995 Salmonella enterica subsp. enterica serovar Indiana str. ATCC 51959 fig|1123516.3.peg.1267 Hydrogenovibrio halophilus DSM 15072 fig|1981674.3.peg.1814 Pseudomonas sp. R9(2017) fig|1947311.3.peg.2053 Pseudomonas sp. UBA2684 fig|1198309.3.peg.4291 Pseudomonas fluorescens ICMP 11288 fig|715451.3.peg.1743 Alteromonas naphthalenivorans fig|316.285.peg.730 Pseudomonas stutzeri fig|1190606.3.peg.313 Enterovibrio calviensis 1F-211 WP_009176189.1 WP_097050713.1 Thalassospira xiamenensis fig|1208323.3.peg.893 Celeribacter baekdonensis B30 KZK95863.1 Pseudovibrio sp. Ad46 fig|101571.310.peg.3956 Burkholderia ubonensis fig|1882791.3.peg.1790 Burkholderia sp. CF099 fig|1736536.3.peg.4809 Variovorax sp. Root434 PIG30812.1 Janthinobacterium sp. 35 fig|1798244.3.peg.1046 Gallionellales bacterium GWA2_55_18 fig|1131551.3.peg.1124 Methylotenera sp. 1P/1 fig|1843082.3.peg.1574 Macromonas sp. BK-30 fig|279058.16.peg.4721 Collimonas arenae fig|1548123.6.peg.1144 Pusillimonas sp. T2 fig|380394.4.peg.276 Acidithiobacillus ferrooxidans ATCC 53993 WP_080292858.1 fig|101571.162.peg.3605 Burkholderia ubonensis fig|1382803.3.peg.22 Chromobacterium amazonense fig|930.4.peg.3851 Acidithiobacillus thiooxidans fig|1261658.3.peg.1787 Bibersteinia trehalosi Y31 fig|1679001.3.peg.631 Pasteurellaceae bacterium NI1060 fig|1334187.3.peg.653 Haemophilus influenzae KR494 fig|1581107.3.peg.1286 Neisseria sp. HMSC15G01 fig|486.24.peg.152 Neisseria lactamica fig|1953412.3.peg.1956 bacterium UBP10 UBA1160 WP_090322045.1 Nitrosomonas oligotropha fig|2013740.3.peg.1400 Deltaproteobacteria bacterium HGW-Deltaproteobacteria-13 fig|1907413.3.peg.3170 Rhizobium sp. RU33A fig|1817963.3.peg.856 Roseomonas deserti fig|2035448.3.peg.1752 Rhizobium sp. C5 WP_014077019.1 fig|1648404.4.peg.2797 Erythrobacter atlanticus fig|359.11.peg.6331 Agrobacterium rhizogenes fig|887144.4.peg.573 Rhizobium taibaishanense fig|1116389.3.peg.333 Devosia insulae DS-56 fig|121719.10.peg.3421 Pannonibacter phragmitetus fig|34002.6.peg.3570 Paracoccus alcaliphilus fig|1940281.4.peg.1560 Hoeflea sp. fig|1040981.5.peg.1561 Mesorhizobium ciceri WSM4083 fig|410764.3.peg.807 Rhizobium multihospitium fig|1825934.3.peg.3111 Rhizobium anhuiense fig|1952824.3.peg.3061 Rhodobiaceae bacterium UBA3976 fig|1871086.3.peg.2153 Brevundimonas sp. fig|1588932.9.peg.647 Brevundimonas naejangsanensis fig|1951751.3.peg.1538 Erythrobacteraceae bacterium UBA1460 fig|1843368.3.peg.904 Sphingobium sp. RAC03 fig|155892.10.peg.3219 Caulobacter vibrioides fig|43057.4.peg.4537 Rhodobacter azotoformans fig|1514904.3.peg.974 Ahrensia marina fig|1338034.3.peg.722 Vibrio parahaemolyticus O1:Kuk str. FDA R31 fig|150340.18.peg.1837 Vibrio antiquarius fig|196024.5.peg.3821 Aeromonas dhakensis fig|244366.32.peg.1886 Klebsiella variicola fig|180957.35.peg.1654 Pectobacterium brasiliense fig|55601.149.peg.665 Vibrio anguillarum fig|121723.5.peg.2901 Photobacterium sp. SKA34 fig|1584.170.peg.837 Proteus mirabilis fig|40324.136.peg.3276 Stenotrophomonas maltophilia fig|1122188.5.peg.411 Lysobacter spongiicola DSM 21749 fig|2032566.3.peg.2826 Xanthomonadaceae bacterium NML93-0792 fig|287.1731.peg.2578 Pseudomonas aeruginosa fig|251702.3.peg.1529 Pseudomonas syringae pv. antirrhini fig|1960829.3.peg.5912 Pseudomonas sp. MF6394 fig|76759.17.peg.5093 Pseudomonas monteilii fig|1981678.3.peg.5241 Pseudomonas sp. R45(2017) fig|1699620.3.peg.3028 Pseudomonas sp. RIT-PI-r fig|191391.4.peg.2140 Pseudomonas salomonii fig|1844093.4.peg.7190 Pseudomonas sp. 22 E 5 fig|287.1744.peg.1414 Pseudomonas aeruginosa fig|287.1987.peg.910 Pseudomonas aeruginosa fig|287.4372.peg.4481 Pseudomonas aeruginosa fig|1856685.4.peg.2159 Pseudomonas sp. TCU-HL1 fig|1718920.3.peg.3357 Pseudomonas sp. ICMP 8385 fig|1781066.3.peg.2816 Duganella sp. HH101 fig|95485.5.peg.60 Burkholderia stabilis fig|1572871.6.peg.588 Janthinobacterium sp. BJB304 WP_034208069.1 Burkholderia cepacia WP_074283015.1 Burkholderia sp. GAS332 fig|1168169.3.peg.2570 Methylomonas sp. 11b fig|1899355.16.peg.1328 Oceanospirillaceae bacterium WP_093197597.1 Variovorax sp. YR750 fig|1660091.3.peg.1650 Bordetella sp. SCN 67-23 fig|134375.17.peg.4387 Achromobacter sp. fig|1426114.10.peg.1990 Thiomonas arsenitoxydans fig|1947551.3.peg.1903 Stenotrophomonas sp. UBA2302 fig|1914330.4.peg.2242 Salinisphaera sp. fig|1947037.3.peg.890 Pantoea sp. UBA5707 WP_094422719.1 Kosakonia cowanii WP_079496884.1 WP_088126255.1 Enterobacter kobei WP_049614309.1 Yersinia WP_048263135.1 Pectobacterium peruviense WP_040197602.1 Klebsiella pneumoniae fig|669.34.peg.1586 Vibrio harveyi fig|672.219.peg.1032 Vibrio vulnificus fig|670.1028.peg.1775 Vibrio parahaemolyticus WP_065207673.1 Photobacterium phosphoreum fig|1869214.3.peg.2231 Rheinheimera sp. WP_029795910.1 Vibrio parahaemolyticus fig|1191302.3.peg.1081 Vibrio crassostreae 9ZC77 fig|668.70.peg.1192 Aliivibrio fischeri fig|28229.4.peg.4229 Colwellia psychrerythraea fig|1855726.3.peg.270 Burkholderia sp. KK1 fig|1674888.3.peg.829 Burkholderiales bacterium Beta 02 fig|687412.4.peg.1108 Pseudorhodobacter aquimaris WP_092465129.1 Donghicola eburneus fig|1120653.3.peg.5479 Ensifer sp. LC384 fig|121719.5.peg.401 Pannonibacter phragmitetus fig|1798804.3.peg.1597 Rhizobium sp. 58 fig|1946675.3.peg.3089 Kordiimonas sp. UBA4487 fig|36861.5.peg.1400 Thiobacillus denitrificans fig|1115835.3.peg.1003 Methylotenera versatilis 79 fig|1797188.3.peg.1508 Acidobacteria bacterium RIFCSPLOWO2_12_FULL_60_22 fig|57320.3.peg.123 Pseudodesulfovibrio profundus fig|1267534.3.peg.1238 Acidobacteriaceae bacterium KBS 89 fig|1951344.3.peg.527 Acidobacteriaceae bacterium UBA1307 WP_006226461.1 Achromobacter marplatensis fig|1503054.4.peg.5764 Burkholderia stagnalis WP_006159686.1 Cupriavidus basilensis WP_090191767.1 unclassified Duganella fig|539.8.peg.1698 Eikenella corrodens fig|1946925.3.peg.2129 Micavibrio sp. UBA5701 WP_047031309.1 Hoeflea sp. IMCC20628 fig|1946134.3.peg.1092 Brevundimonas sp. UBA6547 WP_093914930.1 Sulfitobacter marinus fig|1862950.3.peg.1234 Rhizobiales bacterium NRL2 fig|1166078.4.peg.1483 Aureimonas phyllosphaerae fig|709015.3.peg.734 Pontibacter actiniarum DSM 19842 WP_092160028.1 Desulfovibrio ferrireducens fig|2026749.3.peg.3364 Ignavibacteriae bacterium WP_033771991.1 Pantoea agglomerans WP_097097099.1 unclassified Enterobacteriaceae (miscellaneous) fig|1444151.3.peg.2733 Escherichia coli 2-177-06_S3_C2 WP_137545672.1 Escherichia coli Retron- Eco3 (Ec73) fig|573.14856.peg.3852 Klebsiella pneumoniae WP_072021595.1 Serratia marcescens fig|29571.3.peg.478 Halomonas subglaciescola WP_004534676.1 WP_095622523.1 Halomonas sp. WRN001 fig|376427.4.peg.3223 Halomonas gudaonensis fig|862908.3.peg.745 Halobacteriovorax marinus SJ SCJ40239.1 uncultured Clostridium sp. fig|717962.3.peg.287 Coprococcus catus GD/7 WP_014642259.1 Halobacillus halophilus fig|2009042.3.peg.2106 Pseudomonas sp. Irchel 3H7 fig|1981718.3.peg.4346 Pseudomonas sp. B39(2017) fig|665135.13.peg.1401 Pseudomonas sp. In5 fig|1949067.3.peg.5629 Pseudomonas sp. PICF141 WP_007948552.1 Pseudomonas sp. GM21 SFB61662.1 Delftia tsuruhatensis WP_011615687.1 WP_014778098.1 fig|1429083.4.peg.2612 Pseudomonas hussainii WP_095024014.1 Pseudomonas WP_090203690.1 Pseudomonas asplenii fig|1564423.8.peg.1646 Pseudomonas tolaasii NCPPB 2192 WP_078802277.1 Pseudomonas fluorescens WP_090453229.1 Pseudomonas fig|1306420.5.peg.1032 Burkholderia pseudomallei MSHR5848 fig|1357270.3.peg.1923 Pseudomonas syringae UB246 fig|2018067.3.peg.2950 Pseudomonas sp. FDAARGOS_380 fig|317.311.peg.3241 Pseudomonas syringae fig|287.2309.peg.126 Pseudomonas aeruginosa WP_039522442.1 Pectobacterium brasiliense WP_080861357.1 Klebsiella pneumoniae WP_014542745.1 Erwinia sp. Ejp617 OSL25696.1 Escherichia coli TA255 fig|1125693.3.peg.761 Proteus mirabilis WGLW4 KMK80587.1 Pectobacterium atrosepticum ICMP 1526 fig|550.717.peg.2037 Enterobacter cloacae ACS86154.1 Dickeya paradisiaca Ech703 WP_050122514.1 Yersinia frederiksenii WP_081334048.1 Alteromonas macleodii WP_055016254.1 Pseudoalteromonas sp. P1-13-1a fig|56799.5.peg.478 Colwellia sp. fig|666.3375.peg.2486 Vibrio cholerae PIW62005.1 Shewanella sp. CG12_big_fil_rev_8_21_14_0_65_47_15 OCA54994.1 Photorhabdus namnaonensis CNK75559.1 Yersinia frederiksenii WP_024248662.1 Escherichia WP_088618141.1 Methylovulum psychrotolerans WP_051669880.1 PCJ98666.1 Alteromonadaceae bacterium WP_081919471.1 Acidithiobacillus ferrivorans WP_055769167.1 Stenotrophomonas WP_039422954.1 Xanthomonas vesicatoria WP_078568253.1 Xanthomonas campestris WP_093486747.1 unclassified Pseudoxanthomonas WP_077445058.1 Rhodanobacter sp. C05 WP_092576562.1 Achromobacter sp. NFACC18-2 fig|1330528.3.peg.2198 Escherichia coli NCCP 15656 fig|83655.67.peg.2965 Leclercia adecarboxylata fig|573.10044.peg.2850 Klebsiella pneumoniae WP_071888955.1 Enterobacterales fig|1799789.3.peg.4357 Paraglaciecola hydrolytica fig|2024839.8.peg.1563 Marinovum sp. fig|1381081.7.peg.1167 Vibrio panuliri fig|670.908.peg.3444 Vibrio parahaemolyticus fig|626887.3.peg.2431 Marinobacter nanhaiticus D15-8W fig|1913989.101.peg.1616 Gammaproteobacteria bacterium fig|262489.9.peg.2938 delta proteobacterium MLMS-1 fig|12035207.3.peg.545 Janthinobacterium sp. 67 fig|28095.13.peg.1040 Burkholderia gladioli fig|941449.3.peg.1262 Desulfovibrio sp. X2 fig|1768806.3.peg.778 Rhodospirillaceae bacterium CCH5-H10 WP_083634830.1 Desulfovibrio sp. DV fig|1231.4.peg.574 Nitrosospira multiformis fig|604089.3.peg.1142 Flavobacterium sinopsychrotolerans fig|357523.3.peg.1851 Flavobacterium sp. 11 fig|1423323.5.peg.321 Flavobacterium sp. AED fig|178356.3.peg.502 Flavobacterium xinjiangense fig|1946545.3.peg.3457 Flavobacterium sp. UBA4120 fig|150146.3.peg.2822 Flavobacterium gillisiae fig|229203.4.peg.1981 Flavobacterium degerlachei fig|280093.5.peg.432 Flavobacterium granuli fig|728056.4.peg.1154 Flavobacterium oncorhynchi fig|143224.8.peg.2343 Zobellia uliginosa fig|1225176.3.peg.4300 Cecembia lonarensis LW9 fig|1434700.3.peg.581 Moheibacter sediminis fig|996.47.peg.468 Flavobacterium columnare fig|172045.56.peg.2231 Elizabethkingia miricola fig|12024823.3.peg.2086 Altibacter sp. fig|2026728.18.peg.4090 Crocinitomicaceae bacterium fig|980584.3.peg.2930 Aquimarina agarivorans fig|1946744.3.peg.1682 Leeuwenhoekiella sp. UBA1003 fig|1046627.3.peg.2526 Bizionia argentinensis JUB59 fig|906888.15.peg.37 Nonlabens ulvanivorans fig|407022.4.peg.2865 Olivibacter domesticus fig|1500282.3.peg.3713 Chryseobacterium sp. CF365 WP_084550290.1 Chryseobacterium scophthalmum fig|190304.8.peg.741 Fusobacterium nucleatum subsp. nucleatum ATCC 25586 fig|1352.1731.peg.603 Enterococcus faecium fig|1428.658.peg.666 Bacillus thuringiensis fig|1497681.3.peg.3095 Listeria newyorkensis fig|1396.1440.peg.4237 Bacillus cereus fig|1917876.3.peg.2997 Blautia sp. Marseille-P3087 fig|1952168.3.peg.215 Lachnospiraceae bacterium UBA7480 fig|1907659.3.peg.1085 Blautia sp. Marseille-P3201T fig|1265309.16.peg.461 Epibacterium mobile F1926 fig|853.163.peg.215 Faecalibacterium prausnitzii fig|1264.5.peg.4 Ruminococcus albus fig|1500289.3.peg.4469 Chryseobacterium sp. OV705 fig|1197728.3.peg.2386 Prevotella conceptionensis 9403948 fig|1947486.3.peg.2515 Sphingobacterium sp. UBA1897 fig|529.12.peg.1303 Ochrobactrum anthropi fig|1523429.3.peg.2936 Rhizobium sp. AAP116 fig|1761878.3.peg.469 Paenibacillus sp. cl6col fig|1462996.4.peg.2634 Paenibacillus yonginensis fig|582475.4.peg.4724 Lysinibacillus xylanilyticus fig|1773.7915.peg.7638 Mycobacterium tuberculosis fig|360310.3.peg.4853 Bacillus sp. CDB3 fig|1396.515.peg.2936 Bacillus cereus fig|662367.4.peg.242 Spirosoma endophyticum fig|1895719.3.peg.2950 Bacteroidales bacterium 45-6 fig|906888.9.peg.926 Nonlabens ulvanivorans fig|694433.3.peg.2346 Saprospira grandis DSM 2844 fig|1167006.5.peg.2941 Desulfocapsa sulfexigens DSM 10523 fig|649724.3.peg.304 Clostridium sp. ATCC BAA-442 fig|1505.32.peg.2959 Paeniclostridium sordellii fig|1953142.3.peg.1858 Bacteroidetes bacterium UBA1947 fig|2029590.3.peg.2754 Mucilaginibacter sp. MD40 fig|29581.33.peg.2300 Janthinobacterium lividum fig|40324.292.peg.236 Stenotrophomonas maltophilia fig|1403329.3.peg.287 Listeria monocytogenes Lm25180 fig|1121865.3.peg.1262 Enterococcus columbae DSM 7374 = ATCC 51263 fig|1120746.3.peg.3113 bacterium MS4 fig|1952299.3.peg.221 Ruminococcaceae bacterium UBA2656 fig|1965604.3.peg.686 Anaeromassilibacillus sp. An250 fig|1673717.3.peg.805 Anaeromassilibacillus senegalensis WP_116884683.1 Victivallis vadensis fig|1948697.3.peg.196 Lentisphaeria bacterium UBA4640 fig|1232460.3.peg.46 Clostridiales bacterium VE202-28 WP_007864340.1 Clostridiales WP_055649738.1 Hungatella hathewayi fig|1226325.3.peg.2005 Clostridium sp. KLE 1755 fig|1432052.10.peg.3166 Eisenbergiella tayi fig|208479.8.peg.4376 Enterocloster bolteae fig|1298920.3.peg.1959 [ Desulfotomaculum ] guttoideum DSM 4024 fig|1776047.3.peg.4241 Clostridium sp. C105KSO15 fig|1946596.3.peg.2399 Hungatella sp. UBA4396 fig|1946603.3.peg.924 Hungatella sp. UBA7603 fig|1410651.3.peg.407 [ Clostridium ] aerotolerans DSM 5434 fig|1697784.3.peg.9617 Clostridia bacterium UC5.1-1D4 fig|1745713.3.peg.3865 Bariatricus massiliensis fig|180332.3.peg.1515 Robinsoniella peoriensis WP_072851604.1 Lactonifactor longoviformis WP_003507561.1 Clostridiales fig|1111728.3.peg.587 Budvicia aquatica DSM 5075 = ATCC 35567 fig|1122977.4.peg.2473 Pragia fontium DSM 5563 = ATCC 49100 fig|1950915.3.peg.189 Clostridiales bacterium UBA644 fig|1950927.3.peg.912 Clostridiales bacterium UBA7187 ERK60856.1 Oscillibacter sp. KLE 1728 WP_009260579.1 Flavonifractor plautii fig|1235797.3.peg.2409 Oscillibacter sp. 1-3 fig|1520815.3.peg.1262 Ruminococcaceae bacterium D5 fig|1855302.3.peg.1138 Pseudobutyrivibrio sp. JW11 fig|43305.5.peg.3631 Butyrivibrio proteoclasticus fig|411463.15.peg.1791 Eubacterium ventriosum ATCC 27560 fig|1235792.3.peg.3837 Lachnospiraceae bacterium M18-1 fig|97139.3.peg.669 Schaedlerella arabinosiphila fig|1291051.3.peg.1165 Mediterraneibacter glycyrrhizinilyticus JCM 13369 fig|1532.6.peg.4793 Blautia coccoides fig|1121114.4.peg.5478 Blautia producta ATCC 27340 = DSM 2950 fig|1262776.3.peg.1908 Clostridium sp. CAG:149 fig|1262792.3.peg.1164 Clostridium sp. CAG:299 fig|1262995.3.peg.2852 Firmicutes bacterium CAG:646 fig|537007.17.peg.3146 Blautia hansenii DSM 20583 fig|1965569.3.peg.1928 Lachnoclostridium sp. An169 fig|1952411.3.peg.2018 Ruminococcaceae bacterium UBA6353 fig|1965578.3.peg.1947 Pseudoflavonifractor sp. An187 WP_001775049.1 Escherichia coli Retron- Eco5 (Ec107) WP_012602583.1 WP_015962464.1 Enterobacteriaceae bacterium strain FGI 57 fig|1005999.3.peg.3342 Leminorella grimontii ATCC 33999 = DSM 5078 fig|1378073.3.peg.795 Enterobacter sp. CC120223-11 fig|911023.3.peg.138 Yokenella regensburgei ATCC 49455 fig|1834193.3.peg.4113 Enterococcus sp. 9E7_DIV0242 fig|1649188.10.peg.406 Listeria goaensis fig|1430899.3.peg.278 Listeria fleischmannii 1991 fig|1211844.4.peg.748 Candidatus Stoquefichus massiliensis AP9 fig|1658109.3.peg.34 Candidatus Stoquefichus sp. SB1 fig|1262793.3.peg.950 Clostridium sp. CAG:302 fig|1262908.3.peg.1120 Mycoplasma sp. CAG:956 fig|1674844.3.peg.242 Clostridiales bacterium Firm_06 fig|1410672.3.peg.2823 Ruminococcus flavefaciens ND2009 fig|1947424.3.peg.1718 Ruminococcus sp. UBA4310 fig|1265.9.peg.2602 Ruminococcus flavefaciens fig|1336236.3.peg.1817 Ruminococcus flavefaciens ATCC 19208 CDC65895.1 Ruminococcus sp. CAG:57 WP_092946213.1 Ruminococcaceae bacterium YRB3002 fig|1307.1644.peg.1532 Streptococcus suis WP_050516365.1 Escherichia coli WP_097505494.1 Escherichia coli Retron- Eco6 (Ec48) fig|573.15585.peg.2343 Klebsiella pneumoniae WP_023581669.1 Proteus hauseri WP_079656969.1 Serratia marcescens WP_090085157.1 Phytobacter sp. SCO41 fig|573.15584.peg.1543 Klebsiella pneumoniae WP_023330997.1 Enterobacter cloacae complex fig|72407.673.peg.2552 Klebsiella pneumoniae subsp. pneumoniae CNM01182.1 Yersinia pseudotuberculosis CNG88012.1 Yersinia enterocolitica fig|1925763.3.peg.649 Marinobacter salexigens PKW24121.1 Marinobacter sp. LV10R510-8 WP_045597342.1 Vibrio vulnificus WP_098972386.1 Aeromonas sp. CU5 WP_005172873.1 Yersinia enterocolitica WP_052979504.1 Enterobacteriaceae WP_083069261.1 Pantoea vagans WP_053911905.1 Pseudoalteromonas sp. SW0106-04 fig|1916082.18.peg.39 Alteromonadaceae bacterium WP_046555216.1 Arsukibacterium sp. MJ3 KPW01986.1 Pseudoalteromonas sp. P1-8 WP_094277737.1 Oceanimonas baumannii fig|1414654.3.peg.2005 Oceanisphaera psychrotolerans WP_008133621.1 unclassified Pseudoalteromonas KQA22543.1 Vibrio metoecus WP_000284440.1 Vibrio cholerae WP_011261677.1 Aliivibrio fischeri KEE40622.1 WP_012982829.1 ALL66139.1 Paraburkholderia caribensis MBA4 WP_093223969.1 Pseudomonas vancouverensis fig|12015553.3.peg.2940 Pseudomonas sp. PGPPP1 WP_096082869.1 Pseudomonas aeruginosa ONM67687.1 Pseudomonas aeruginosa fig|316.213.peg.2906 Pseudomonas stutzeri WP_078734267.1 Pseudomonas fluorescens WP_079384669.1 Pseudomonas aeruginosa WP_095948157.1 Variovorax boronicumulans WP_011625020.1 Shewanella sp. MR-7 WP_100292553.1 Aeromonas cavernicola WP_055021484.1 Pseudoalteromonas sp. P1-26 PHS01491.1 Oceanobacter sp. fig|2024618.3.peg.1141 Acinetobacter sp. BS1 WP_114139108.1 Klebsiella pneumoniae WP_077749737.1 Pseudomonas sp. FSL W5-0299 WP_078451378.1 Pseudomonas aeruginosa WP_007245785.1 Pseudomonas syringae group fig|316.280.peg.1454 Pseudomonas stutzeri WP_086822222.1 Pseudomonas aeruginosa WP_073268605.1 Pseudomonas punonensis WP_095280108.1 Lelliottia jeotgali WP_095715328.1 Citrobacter sp. TSA-1 WP_050111525.1 Yersinia WP_013724211.1 Aeromonas veronii WP_021140819.1 Aeromonas salmonicida fig|1094342.5.peg.1611 Alcanivorax xenomutans fig|1932666.4.peg.1886 Haliea sp. WP_087148323.1 Crenothrix polyspora WP_064022638.1 Methylomonas sp. DH-1 PIY64876.1 Shewanella sp. CG_4_10_14_0_8_um_filter_42_13 WP_006710190.1 Vibrio ichthyoenteri WP_045040928.1 Photobacterium iliopiscarium WP_054543201.1 Vibrio splendidus WP_080540293.1 Vibrio vulnificus fig|2032624.3.peg.2540 Halomonas sp. WN018 KJT50308.1 Salmonella enterica subsp. Retron- enterica serovar Heidelberg str. Sen1 RI-11-014588 (Se72) WP_005761319.1 ODQ05744.1 Shigella sp. FC130 KKW01006.1 Candidatus Saccharibacteria bacterium GW2011_GWC2_48_9 KMZ12260.1 Candidatus Burkholderia humilis SFQ04394.1 Ralstonia sp. NFACC01 WP_025373922.1 Advenella mimigardefordensis WP_093341200.1 Variovorax sp. PDC80 WP_091453700.1 Giesbergeria anulus SAY51889.1 Neisseria weaveri WP_065255232.1 Moraxella lacunata WP_049330876.1 Neisseria fig|1196095.197.peg.151 Gilliamella apicola WP_072956843.1 Vibrio gazogenes fig|857087.3.peg.3286 Methylomonas methanica MC09 fig|1952222.3.peg.1307 Methylococcaceae bacterium UBA3127 WP_039486261.1 Vibrio sinaloensis WP_065545234.1 Vibrio scophthalmi WP_033094845.1 Colwellia psychrerythraea WP_057552475.1 Vibrio cholerae WP_004726393.1 Vibrio furnissii fig|2020862.3.peg.1934 Halobacteriovorax sp. fig|624.1260.peg.1437 Shigella sonnei WP_011516221.1 Burkholderiales fig|1947370.3.peg.1923 Pusillimonas sp. UBA4517 WP_038400955.1 Yersinia pseudotuberculosis fig|1951903.3.peg.117 Halieaceae bacterium UBA3099 WP_024914507.1 Chania multitudinisentens WP_042893228.1 Enterobacteriaceae WP_038238211.1 Xenorhabdus szentirmaii EXI65661.1 Candidatus Accumulibacter sp. SK-12 WP_016452106.1 Delftia WP_013517170.1 Alicycliphilus denitrificans OXC73828.1 Caballeronia sordidicola AIO65205.1 Burkholderia oklahomensis WP_013234866.1 Herbaspirillum seropedicae WP_082884385.1 Piscirickettsiaceae bacterium NZ-RLO1 fig|2006849.4.peg.371 Xanthomonadales bacterium WP_074262787.1 Paraburkholderia phenazinium WP_009906786.1 Burkholderia thailandensis WP_022524328.1 WP_081817450.1 Halomonas sp. HL-48 WP_020312233.1 Pseudomonas syringae KPY75916.1 Pseudomonas amygdali pv. tabaci fig|1793966.3.peg.180 Pseudomonas fluvialis fig|1891229.16.peg.2033 Pseudomonadales bacterium WP_099454886.1 Pseudomonas putida WP_092400423.1 Pseudomonas sp. NFACC39-1 WP_012315430.1 Pseudomonas putida WP_020799819.1 Pseudomonas sp. G5(2012) WP_004574016.1 fig|1435425.3.peg.787 Pseudomonas sp. QTF5 WP_045490543.1 Pseudomonas sp. StFLB209 WP_011506503.1 Chromohalobacter salexigens fig|1609967.3.peg.3047 Halomonas sp. HG01 fig|1492738.3.peg.2698 Flavobacterium seoulense WP_092849245.1 Algibacter pectinivorans WP_025835957.1 Bacteroides fig|2025877.3.peg.668 Parabacteroides sp. AT13 fig|246787.6.peg.2081 Bacteroides cellulosilyticus fig|1339287.3.peg.1113 Bacteroides fragilis str. 3986 T (B) 9 fig|1946017.3.peg.1516 Alistipes sp. UBA940 WP_038655380.1 Mucinivorans hirudinis WP_093669272.1 Tenacibaculum sp. MAR_2009_124 WP_073241067.1 Flavobacterium flevense WP_096193803.1 Cytophagales bacterium TFI 002 WP_076357635.1 WP_073238193.1 Pedobacter caeni WP_076451370.1 WP_091906542.1 Porphyromonadaceae bacterium KH3R12 WP_051365712.1 Flavobacterium saliperosum fig|1938609.3.peg.1765 Flavobacterium sp. LM4 SDJ72221.1 Flavobacterium noncentrifugens fig|1985174.3.peg.2584 Chitinophagaceae bacterium IBVUCB2 WP_092737749.1 Riemerella columbipharyngis fig|192149.3.peg.42 Muricauda sp. fig|418630.3.peg.1685 Rhodobacter megalophilus fig|1915314.3.peg.3469 Thioclava sp. DLFJ5-1 fig|2030815.3.peg.2725 Marinosulfonomonas sp. fig|2035451.3.peg.4632 Rhizobium sp. L18 WP_043872258.1 Celeribacter indicus WP_055683826.1 Jannaschia rubra fig|1947537.3.peg.498 Sphingopyxis sp. UBA6198 WP_069065961.1 Sphingobium sp. RAC03 WP_084280100.1 Novosphingobium sp. B1 fig|1895845.3.peg.487 Sphingobium sp. 66-54 GAK73419.1 Agrobacterium rubi TR3 = NBRC 13261 WP_090966398.1 Aureimonas phyllosphaerae WP_091860144.1 Bosea robiniae WP_085092006.1 Azospirillum oryzae fig|1528100.4.peg.28 Methylomagnum ishizawai fig|132057.3.peg.9515 Calothrix sp. PCC 7103 fig|103690.10.peg.3571 Nostoc sp. PCC 7120 = FACHB-418 fig|1137095.11.peg.15 Scytonema sp. HK-05 CDZ48826.1 Neorhizobium galegae bv. officinalis WP_072340070.1 Devosia enhydra OYR18277.1 Ochrobactrum thiophenivorans WP_093509439.1 Sphingopyxis sp. YR583 WP_081799025.1 Novosphingobium resinovorum PIY55545.1 Zetaproteobacteria bacterium CG_4_10_14_0_8_um_filter_49_80 SDT44912.1 Bradyrhizobium canariense WP_096350346.1 WP_074962594.1 Jannaschia rubra WP_038724888.1 Burkholderia pseudomallei WP_012217410.1 Burkholderia multivorans WP_100428762.1 Janthinobacterium sp. 67 WP_082161008.1 Candidatus Competibacter denitrificans AFL73219.1 Thiocystis violascens DSM 198 WP_014427842.1 fig|364030.3.peg.3554 Thiomonas delicata KGW20495.1 Burkholderia pseudomallei MSHR2451 SFE83076.1 Variovorax sp. OK212 WP_013028226.1 Sideroxydans lithotrophicus WP_080311424.1 Burkholderia pseudomallei fig|337.13.peg.3872 Burkholderia glumae WP_082643860.1 Pseudomonas CKH90039.1 Pseudomonas aeruginosa WP_083287254.1 unclassified Janthinobacterium WP_122648546.1 Burkholderia pseudomallei WP_082706753.1 unclassified Pseudomonas WP_080936076.1 Klebsiella pneumoniae WP_000746343.1 Enterobacteriaceae EMX54653.1 Escherichia coli MP020980.2 WP_053270700.1 Escherichia coli fig|1736224.3.peg.3731 Serratia sp. Leaf51 fig|1175299.4.peg.709 Dickeya zeae ZJU1202 WP_001461245.1 Enterobacteriaceae fig|617145.3.peg.3535 Vibrio splendidus 1F-157 fig|1440054.3.peg.3851 Vibrio sp. OY15 fig|617135.3.peg.594 Aliivibrio fischeri ZF-211 WP_023267764.1 Shewanella decolorationis fig|1481663.36.peg.3628 Vibrio metoecus fig|670.893.peg.2716 Vibrio parahaemolyticus fig|680.33.peg.5391 Vibrio campbellii fig|298386.8.peg.4344 Photobacterium profundum SS9 fig|663.73.peg.714 Vibrio alginolyticus fig|1333511.3.peg.3208 Pseudoalteromonas haloplanktis TAB23 WP_064574154.1 Hafnia paralvei WP_064645509.1 Obesumbacterium proteus fig|630.105.peg.4248 Yersinia enterocolitica fig|1400673.7.peg.1969 Legionella pneumophila str. Corby WP_092678546.1 Rosenbergiella nectarea WP_069476513.1 Raoultella ornithinolytica fig|1267535.3.peg.2394 Bryobacterales bacterium KBS 96 WP_000446053.1 Acinetobacter baumannii fig|1948587.3.peg.786 Gammaproteobacteria bacterium UBA1902 WP_014949305.1 Alteromonas macleodii fig|1797397.3.peg.2386 Bdellovibrionales bacterium RIFOXYC1_FULL_54_43 fig|1386968.3.peg.847 Francisella tularensis subsp. novicida PA10-7858 WP_074900850.1 fig|1975705.3.peg.898 Psychrobacter sp. FDAARGOS_221 WP_066184577.1 Arcobacter fig|1780380.4.peg.4010 Eubacteriaceae bacterium CHKCI004 fig|556261.3.peg.2546 Clostridium sp. D5 fig|1193534.6.peg.2375 uncultured Flavonifractor sp. fig|1042163.3.peg.3771 Brevibacillus laterosporus LMG 15441 WP_062492190.1 Paenibacillus sp. 320-W WP_081674606.1 Lactobacillus harbinensis WP_050781686.1 Lactobacillus coryniformis WP_021109137.1 Enterococcus faecium WP_046309803.1 Staphylococcus CBL03706.1 Gordonibacter pamelaeae 7-10-1-b WP_090944285.1 Pelosinus propionicus WP_077305443.1 Clostridium beijerinckii fig|410072.5.peg.40 Coprococcus comes WP_011669870.1 Leptospira borgpetersenii WP_015565235.1 Faecalibacterium prausnitzii CUO23478.1 Faecalibacterium prausnitzii WP_085748688.1 Rhizobacter gummiphilus WP_093270014.1 Psychrobacillus sp. OK032 SHE86352.1 Atopostipes suicloacalis DSM 15692 WP_000346292.1 unclassified Streptococcus WP_080465410.1 Lactobacillus plantarum WP_080662531.1 Lactobacillus brevis WP_093131554.1 Salinibacillus kushneri WP_093336905.1 Salibacterium halotolerans fig|1974627.3.peg.386 Candidatus Levybacteria bacterium CG_4_9_14_0_2_um_filter_35_21 fig|1802603.3.peg.453 Candidatus Woykebacteria bacterium RIFCSPHIGHO2_12_FULL_45_10 fig|392734.5.peg.3006 Terriglobus roseus AGL61879.1 Candidatus Saccharimonas aalborgensis fig|319224.16.peg.2726 Shewanella putrefaciens CN-32 fig|1720343.3.peg.1263 Pseudoalteromonas sp. 1_2015MBL_MicDiv fig|1136158.3.peg.3691 Vibrio cyclitrophicus 1F97 fig|666.3017.peg.1000 Vibrio cholerae fig|1909458.3.peg.2277 Salinivibrio sp. ML198 fig|1638949.3.peg.831 Vibrio sp. ECSMB14106 fig|1493915.3.peg.158 Pseudoalteromonas sp. NJ631 BAC94535.1 Vibrio vulnificus YJ016 fig|1191313.3.peg.1135 Vibrio splendidus 1S-124 fig|670.1244.peg.3807 Vibrio parahaemolyticus fig|1659714.3.peg.4264 Citrobacter braakii fig|1192730.4.peg.1976 Salmonella enterica subsp. enterica serovar Kintambo fig|550.1216.peg.4296 Enterobacter cloacae WP_072269713.1 Serratia WP_053898075.1 Escherichia coli fig|624.1264.peg.1635 Shigella sonnei fig|1181777.3.peg.78 Escherichia coli KTE233 fig|1802256.3.peg.310 Sulfurimonas sp. RIFOXYB12_FULL_35_9 PHR73342.1 Arcobacter sp. fig|2014260.3.peg.3813 bacterium (Candidatus Blackallbacteria) CG13_big_fil_rev_8_21_14_2_50_49_14 WP_042497590.1 Vibrio maritimus WP_063522799.1 Vibrio sp. HI00D65 WP_004186757.1 Enterobacteriaceae WP_040122746.1 Vibrio WP_086046550.1 Vibrio harveyi group WP_063849005.1 Enterobacter cloacae WP_023486614.1 Enterobacteriaceae WP_070992278.1 Pseudoalteromonas byunsanensis fig|1005665.3.peg.2532 Kosakonia oryzendophytica fig|1219066.3.peg.3636 Vibrio parahaemolyticus NBRC 12711 fig|1225184.4.peg.1222 Pantoea sp. A4 fig|675814.3.peg.1256 Vibrio coralliilyticus ATCC BAA-450 SFR59865.1 Pseudobutyrivibrio sp. NOR37 fig|853.16.peg.1112 Faecalibacterium prausnitzii fig|1965572.3.peg.1423 Pseudoflavonifractor sp. An176 fig|588581.3.peg.3589 Ruminiclostridium papyrosolvens DSM 2782 fig|1396.1409.peg.4169 Bacillus cereus fig|1428.538.peg.4047 Bacillus thuringiensis fig|1465.16.peg.946 Brevibacillus laterosporus WP_087385137.1 AIF42417.1 Virgibacillus sp. SK37 WP_076543941.1 Halanaerobium kushneri fig|1121093.3.peg.3089 Bacillus panaciterrae DSM 19096 fig|29367.3.peg.2029 Clostridium puniceum WP_089719707.1 Halanaerobium congolense fig|307249.3.peg.3585 uncultured Sporomusa sp. WP_072949666.1 Ruminococcus flavefaciens CCX81854.1 Ruminococcus sp. CAG:108 fig|1491.669.peg.2217 Clostridium botulinum fig|1872455.3.peg.401 Alkaliphilus sp. fig|576117.5.peg.4005 Celeribacter halophilus fig|1225647.3.peg.1829 Phaeobacter sp. 11ANDIMAR09 fig|1380380.4.peg.1574 Ahrensia sp. 13_GOM-1096m fig|293.7.peg.2956 Brevundimonas diminuta WP_095437634.1 Rhizobium sp. 11515TR fig|1912891.7.peg.702 Sphingobium sp. fig|1736574.3.peg.4024 Pseudoxanthomonas sp. Root630 fig|227946.13.peg.4105 Xanthomonas translucens pv. poae fig|1761791.3.peg.4793 Lysobacter sp. yr284 fig|1560195.5.peg.485 Janthinobacterium sp. BJB301 fig|1503054.43.peg.6257 Burkholderia stagnalis fig|1207504.10.peg.4279 Burkholderia pseudomultivorans WP_092172515.1 unclassified Pseudomonas WP_074815429.1 Pseudomonas syringae fig|150146.3.peg.3162 Flavobacterium gillisiae fig|76832.8.peg.3775 Myroides odoratimimus fig|1202724.3.peg.994 Flavobacterium akiainvivens fig|1805473.3.peg.3678 Chryseobacterium timonianum fig|253.33.peg.3826 Chryseobacterium indologenes WP_076561634.1 Chryseobacterium indoltheticum fig|12024823.3.peg.95 Altibacter sp. fig|1250278.4.peg.3462 Salegentibacter sp. Hel I 6 fig|1797342.3.peg.689 Bacteroidetes bacterium GWF2_33_38 WP_084184261.1 Chryseobacterium ureilyticum fig|1948560.3.peg.3003 Deltaproteobacteria bacterium UBA6106 fig|1392.364.peg.2564 Bacillus anthracis fig|872970.3.peg.1713 Amphibacillus marinus fig|1385514.3.peg.313 Pontibacillus yanchengensis Y32 fig|76853.4.peg.2614 Solibacillus silvestris fig|1423774.3.peg.1262 Lactobacillus nantensis DSM 16982 fig|1410670.3.peg.2844 Ruminococcus flavefaciens MA2007 fig|169435.7.peg.1348 Anaerotruncus colihominis fig|1946597.3.peg.2104 Hungatella sp. UBA4568 fig|1948087.3.peg.796 Firmicutes bacterium UBA6113 fig|642492.3.peg.2638 Cellulosilyticum lentocellum DSM 5427 fig|1950841.3.peg.2383 Clostridiales bacterium UBA2436 fig|1555512.3.peg.1251 Salipiger marinus fig|383381.3.peg.2538 Erythrobacter sp. JL475 WP_081629462.1 fig|1736258.3.peg.3392 Methylobacterium sp. Leaf112 fig|1950192.3.peg.426 Anaerolineales bacterium UBA2232 fig|170623.6.peg.4661 Azotobacter beijerinckii fig|170623.7.peg.704 Azotobacter beijerinckii fig|1981099.3.peg.513 Niveispirillum lacus fig|1250539.3.peg.3491 Pelagibaca abyssi fig|1947582.3.peg.2979 Sulfitobacter sp. UBA1132 fig|1909294.17.peg.3456 Rhizobiales bacterium fig|1735583.3.peg.1657 Pseudovibrio sp. W64 fig|670.1220.peg.4688 Vibrio parahaemolyticus fig|1004786.3.peg.925 Alteromonas mediterranea DE1 fig|2013797.3.peg.2109 Gammaproteobacteria bacterium HGW-Gammaproteobacteria-15 fig|1948580.3.peg.3400 Gammaproteobacteria bacterium UBA1012 fig|1714300.3.peg.306 Marinobacterium profundum fig|1961547.3.peg.1371 Desulfobulbaceae bacterium UBA2273 fig|441162.10.peg.6621 Burkholderia oklahomensis C6786 fig|615.307.peg.4666 Serratia marcescens fig|631.3.peg.1883 Yersinia intermedia fig|1763535.3.peg.1547 Hydrogenophaga crassostreae fig|43263.5.peg.2702 Pseudomonas alcaligenes fig|244366.46.peg.3595 Klebsiella variicola fig|1224150.8.peg.3856 Dickeya paradisiaca NCPPB 2511 fig|61645.10.peg.2019 Enterobacter asburiae fig|1948706.3.peg.2225 Opitutae bacterium UBA1333 fig|2026771.13.peg.1697 Opitutae bacterium fig|2026771.11.peg.1955 Opitutae bacterium fig|2026772.5.peg.424 Opitutales bacterium fig|2026801.20.peg.1798 Verrucomicrobiales bacterium fig|2026801.14.peg.1176 Verrucomicrobiales bacterium fig|1951369.3.peg.1157 Akkermansiaceae bacterium UBA6946 fig|1977087.12.peg.1918 Proteobacteria bacterium fig|12026779.14.peg.4171 Planctomycetaceae bacterium fig|12026779.28.peg.3264 Planctomycetaceae bacterium fig|12026779.30.peg.3181 Planctomycetaceae bacterium fig|2026779.29.peg.2310 Planctomycetaceae bacterium fig|1797235.3.peg.3 Acinetobacter sp. RIFCSPHIGHO2_12_41_5 fig|316.284.peg.937 Pseudomonas stutzeri fig|296.11.peg.442 Pseudomonas fragi fig|1981714.3.peg.993 Pseudomonas sp. B5(2017) fig|150340.44.peg.6020 Pseudomonas fuscovaginae fig|1761897.3.peg.509 Pseudomonas sp. ok272 fig|1402514.3.peg.154 Pseudomonas aeruginosa BWHPSA014 fig|1938440.3.peg.5997 Pseudomonas sp. T fig|1566250.3.peg.959 Pseudomonas sp. NFACC02 fig|316.357.peg.479 Pseudomonas stutzeri fig|287.4433.peg.2945 Pseudomonas aeruginosa fig|1970515.3.peg.709 Hydrogenophilales bacterium 12-61-10 fig|95486.85.peg.1748 Burkholderia cenocepacia fig|292.61.peg.8104 Burkholderia cepacia fig|1408450.3.peg.3766 Methylobacter tundripaludum 21/22 fig|157910.3.peg.5727 Paraburkholderia tuberum fig|279058.16.peg.4239 Collimonas arenae fig|1537272.3.peg.1916 Janthinobacterium sp. HH100 fig|1218081.3.peg.1751 Paraburkholderia kururiensis subsp. thiooxydans NBRC 107107 fig|573.14059.peg.3113 Klebsiella pneumoniae fig|40324.192.peg.51 Stenotrophomonas maltophilia fig|1219041.3.peg.4613 Sphingomonas azotifigens NBRC 15497 fig|1561196.3.peg.560 Burkholderia sp. E7m39 fig|1882750.3.peg.1035 Burkholderia sp. GAS332 fig|1736266.3.peg.1145 Duganella sp. Leaf126 fig|2015350.3.peg.1640 Burkholderia sp. AU18528 fig|58133.4.peg.815 Nitrosospira sp. NpAV fig|1691980.3.peg.1912 Rhodocyclaceae bacterium Paddy-1 fig|305.393.peg.1023 Ralstonia solanacearum fig|56449.3.peg.3604 Xanthomonas bromi fig|1281282.5.peg.1894 Xanthomonas campestris pv. campestris str. CN14 fig|40324.334.peg.1103 Stenotrophomonas maltophilia fig|1349793.3.peg.2529 Hydrogenophaga taeniospiralis NBRC 102512 fig|1842727.3.peg.1491 Rhodoferax koreense fig|1619952.3.peg.5158 Burkholderiaceae bacterium 16 fig|1970380.3.peg.1914 Halothiobacillus sp. 14-55-98 fig|2015568.3.peg.2963 Burkholderiales bacterium PBB6 fig|1752215.3.peg.2312 Gammaproteobacteria bacterium Ga0077554 fig|1706231.5.peg.3125 Janthinobacterium sp. CG23_2 fig12013716.3.peg.2169 Betaproteobacteria bacterium HGW- Betaproteobacteria-4 fig|1946997.3.peg.3049 Nitrospira sp. UBA7655 fig|765913.3.peg.2527 Thiorhodococcus drewsii AZ1 fig|1743159.3.peg.1891 Polynucleobacter yangtzensis fig|1597955.3.peg.3923 Limnohabitans sp. DM1 fig|1184267.3.peg.1626 Bdellovibrio exovorus JSS fig|101571.190.peg.3007 Burkholderia ubonensis fig|123899.5.peg.1710 Bordetella trematum fig|463035.3.peg.3900 Bordetella genomosp. 12 fig|1395608.4.peg.211 Bordetella genomosp. 5 fig|1947379.3.peg.2784 Rhodoferax sp. UBA5149 WP_074294985.1 Paraburkholderia phenazinium fig|1324617.3.peg.820 Paraburkholderia aspalathi fig|80868.3.peg.3458 Acidovorax cattleyae fig|1388764.3.peg.1840 Pseudogulbenkiania ferrooxidans EGD-HP2 fig|251747.15.peg.4695 Chromobacterium subtsugae fig|670.1020.peg.382 Vibrio parahaemolyticus fig|1055803.3.peg.1434 Pseudoalteromonas sp. TB51 fig|1201036.3.peg.177 Pseudochrobactrum sp. AO18b fig|1220581.4.peg.1434 Agrobacterium rhizogenes NBRC 13257 fig|398.6.peg.6695 Rhizobium tropici fig|931866.6.peg.8184 Bradyrhizobium ottawaense fig|142585.3.peg.1658 Bradyrhizobium sp. C9 fig|1082933.13.peg.1537 Mesorhizobium amorphae CCNWGS0123 fig|1768789.3.peg.791 Methylobacterium sp. CCH7-A2 fig|1381123.3.peg.3819 Aliihoeflea sp. 2WW fig|1297570.3.peg.1970 Mesorhizobium sp. STM 4661 fig|935546.3.peg.3816 Mesorhizobium loti NZP2037 fig|1128253.3.peg.1960 Bradyrhizobium japonicum CCBAU 15354 fig|1444315.4.peg.3983 Lysobacter capsici AZ78 fig|1185327.3.peg.1608 Xanthomonas axonopodis pv. manihotis str. Xam668 fig|1881043.3.peg.2597 Pseudoxanthomonas sp. GM95 ALN84423.1 Lysobacter capsici fig|56460.15.peg.1977 Xanthomonas vesicatoria fig|1317116.6.peg.2759 Oceanicola sp. 22II-s10i fig|564137.3.peg.4320 Roseicitreum antarcticum fig|1952800.3.peg.3583 Rhodobacteraceae bacterium UBA2553 fig|218673.12.peg.3041 Sulfitobacter dubius fig|1912092.3.peg.2119 Nioella sediminis fig|1736558.3.peg.5006 Ensifer sp. Root558 fig|91360.5.peg.3717 Desulforhopalus singaporensis fig|1948756.3.peg.2576 Spirochaetia bacterium UBA2205 fig|1855322.3.peg.103 Bradyrhizobium sp. Rc3b fig|1437360.11.peg.2429 Bradyrhizobium erythrophlei fig|1871052.3.peg.1026 Afipia sp. fig|1038860.3.peg.8756 Bradyrhizobium elkanii WSM2783 fig|1898112.54.peg.3758 Rhodospirillaceae bacterium fig|1660129.3.peg.4854 Phenylobacterium sp. SCN 70-31 fig|1482074.3.peg.4109 Hartmannibacter diazotrophicus fig|1970306.3.peg.552 Acidocella sp. 35-58-6 fig|1686310.5.peg.1409 Bartonella apis fig|1798192.3.peg.1953 Thalassospira sp. KO164 fig|1235461.17.peg.11 Sinorhizobium meliloti GR4 fig|442.12.peg.222 Gluconobacter oxydans fig|1938607.3.peg.1954 Sphingomonas sp. LM7 fig|1231624.3.peg.39 Asaia bogorensis NBRC 16594 fig|1121271.3.peg.4112 Gemmobacter nectariphilus DSM 15620 fig|33059.16.peg.1690 Acidithiobacillus caldus fig|502025.10.peg.925 Haliangium ochraceum DSM 14365 fig|1734406.3.peg.691 Alphaproteobacteria bacterium BRH_c36 fig|1979207.3.peg.4304 Parvularcula sp. fig|1953057.3.peg.74 Parvularculaceae bacterium UBA4496 fig|858423.3.peg.10004 Bradyrhizobium arachidis fig|1267128.3.peg.2015 Sphingopyxis granuli fig|582667.3.peg.5553 Methylobacterium pseudosasicola fig|1187852.3.peg.2712 Methylobacterium tarhaniae fig|582675.3.peg.1247 Methylobacterium gossipiicola fig|1951640.3.peg.515 Deferribacteraceae bacterium UBA6799 fig|1948417.4.peg.1606 Alphaproteobacteria bacterium UBA6187 fig|45074.5.peg.981 Legionella santicrucis fig|1434232.4.peg.2927 Magnetofaba australis IT-1 fig|1945950.3.peg.3568 Acinetobacter sp. UBA6526 fig|106654.22.peg.994 Acinetobacter nosocomialis fig|1977883.3.peg.3023 Acinetobacter sp. ANC 3903 fig|1945948.3.peg.700 Acinetobacter sp. UBA5984 fig|1226327.3.peg.2796 Acinetobacter kookii fig|1879049.4.peg.5949 Acinetobacter sp. WCHAc010034 fig|1945955.3.peg.1951 Acinetobacter sp. UBA7614 fig|1675530.3.peg.2149 Acinetobacter genomosp. 33YU fig|1310638.3.peg.1006 Acinetobacter baumannii 1437282 fig|1400001.4.peg.34 Necropsobacter massiliensis fig|1132496.5.peg.136 Pasteurella multocida subsp. multocida str. HN06 fig|1908263.4.peg.2604 Rodentibacter trehalosifermentans fig|375432.4.peg.200 Haemophilus influenzae R3021 fig|400668.8.peg.3776 Marinomonas sp. MWYL1 fig|1913989.193.peg.841 Gammaproteobacteria bacterium fig|856793.5.peg.1975 Micavibrio aeruginosavorus ARL-13 SBW23286.1 Citrobacter europaeus fig|1736225.3.peg.985 Erwinia sp. Leaf53 fig|29486.12.peg.818 Yersinia ruckeri fig|914128.3.peg.2502 Serratia symbiotica str. Tucson fig|1796497.3.peg.952 Grimontia celer fig|1095649.3.peg.3298 Vibrio cholerae O1 str. EM-1676A fig|137584.4.peg.1627 Thalassomonas viridans fig|173990.3.peg.1773 Rheinheimera pacifica fig|1720343.3.peg.3189 Pseudoalteromonas sp. 1_2015MBL_MicDiv fig|1202962.4.peg.1481 Moritella marina ATCC 15381 fig|669.50.peg.2993 Vibrio harveyi fig|691.32.peg.1517 Vibrio natriegens fig|156578.3.peg.2521 Alteromonadales bacterium TW-7 fig|661.14.peg.380 Photobacterium angustum fig|654.94.peg.1733 Aeromonas veronii fig|703.9.peg.319 Plesiomonas shigelloides fig|589873.36.peg.1971 Alteromonas australica fig|28107.3.peg.3571 Pseudoalteromonas espejiana fig|1547444.3.peg.4264 Pseudoalteromonas sp. PLSV fig|629266.7.peg.847 Pseudomonas syringae pv. actinidiae str. M302091 fig|251722.19.peg.4059 Pseudomonas amygdali pv. aesculi fig|587851.4.peg.1470 Pseudomonas chlororaphis subsp. aureofaciens fig|1265490.3.peg.2330 Pseudomonas sp. URMO17WK12:I8 fig|316.101.peg.3534 Pseudomonas stutzeri fig|1916993.3.peg.4917 Pseudomonas putida fig|1628833.3.peg.2448 Pseudomonas sp. ES3-33 fig|1283291.4.peg.1991 Pseudomonas sp. URMO17WK12:I11 fig|83963.5.peg.3885 Pseudomonas syringae pv. papulans fig|1206777.3.peg.4334 Pseudomonas sp. Lz4W fig|113268.3.peg.3785 Bathymodiolus platifrons methanotrophic gill symbiont fig|1131284.3.peg.1562 zeta proteobacterium SCGC AB-137-C09 fig|2026807.7.peg.2258 Zetaproteobacteria bacterium fig|281689.4.peg.2060 Desulfuromonas acetoxidans DSM 684 fig|1188231.4.peg.1200 Mariprofundus ferrooxydans M34 fig|1367489.3.peg.682 Aliivibrio fischeri SA1G fig|1873135.3.peg.4249 Shewanella sp. SACH fig|663.73.peg.2465 Vibrio alginolyticus fig|1588629.3.peg.1134 Aeromonas sp. L 1B5 3 fig|1121922.3.peg.3454 Glaciecola pallidula DSM 14239 = ACAM 615 fig|351745.9.peg.2506 Shewanella sp. W3-18-1 fig|29497.20.peg.3798 Vibrio splendidus fig|1367486.3.peg.187 Aliivibrio fischeri CB37 fig|511062.4.peg.1890 Oceanimonas sp. GK1 fig|654.12.peg.188 Aeromonas veronii fig|29497.21.peg.4482 Vibrio splendidus fig|1659713.3.peg.560 Enterobacter bugandensis fig|1124991.3.peg.3617 Morganella morganii subsp. morganii KT fig|104623.3.peg.1381 Serratia sp. ATCC 39006 fig|1256989.3.peg.902 Providencia alcalifaciens R90-1475 fig|1125694.3.peg.1143 Proteus mirabilis WGLW6 fig|574096.6.peg.2693 Pantoea allii fig|1095774.3.peg.2623 Pantoea ananatis PA13 fig|869692.4.peg.2910 Escherichia coli 3003 WP_140159440.1 Escherichia coli Retron- Eco2 (Ec67) fig|550.437.peg.1444 Enterobacter cloacae fig|573.13605.peg.2600 Klebsiella pneumoniae fig|550.285.peg.3783 Enterobacter cloacae fig|1265672.3.peg.3869 Salmonella enterica subsp. enterica serovar Agona str. 70.E.05 fig|573.10028.peg.542 Klebsiella pneumoniae fig|749537.3.peg.218 Escherichia coli MS 115-1 ANK06786.1 Escherichia coli O25b:H4 fig|670.880.peg.975 Vibrio parahaemolyticus fig|1192730.4.peg.3 Salmonella enterica subsp. enterica serovar Kintambo fig|1224144.4.peg.4030 Dickeya sp. CSL RW240 fig|568766.10.peg.2937 Dickeya sp. NCPPB 3274 fig|1076549.3.peg.4260 Pantoea rodasii fig|548.102.peg.3401 Klebsiella aerogenes fig|630.90.peg.1795 Yersinia enterocolitica fig|79883.5.peg.266 Bacillus horikoshii fig|180861.3.peg.3762 Bacillus thuringiensis serovar sumiyoshiensis fig|1390.157.peg.339 Bacillus amyloliquefaciens fig|293386.15.peg.304 Bacillus stratosphericus fig|1053181.3.peg.3820 Bacillus cereus BAG2X1-3 fig|1884375.3.peg.681 Paenibacillus sp. PDC88 fig|1334735.5.peg.923 Sporosarcina koreensis fig|79884.3.peg.1120 Bacillus pseudalcaliphilus fig|1628206.3.peg.4802 Bacillus sp. LK2 fig|1396.1605.peg.6235 Bacillus cereus fig|182710.3.peg.317 Oceanobacillus iheyensis fig|860.10.peg.486 Fusobacterium periodonticum fig|1855308.3.peg.1467 Trichococcus ilyis fig|931626.3.peg.151 Acetobacterium woodii DSM 1030 fig|1965575.3.peg.2547 Lachnoclostridium sp. An181 fig|1352.2757.peg.71 Enterococcus faecium fig|1299895.3.peg.900 Listeria monocytogenes CFSAN002349 fig|53346.29.peg.1591 Enterococcus mundtii fig|1649188.10.peg.1545 Listeria goaensis fig|158847.6.peg.432 Megamonas hypermegale fig|1121289.3.peg.2775 Clostridiisalibacter paucivorans DSM 22131 fig|1950885.3.peg.858 Clostridiales bacterium UBA4693 fig|1965576.3.peg.1978 Pseudoflavonifractor sp. An184 fig|1952416.3.peg.1629 Ruminococcaceae bacterium UBA642 fig|1262803.3.peg.8 Clostridium sp. CAG:413 fig|28037.216.peg.60 Streptococcus mitis fig|1074052.3.peg.33 Streptococcus sobrinus TCI-9 fig|1304.207.peg.1536 Streptococcus salivarius fig|1154859.3.peg.955 Streptococcus agalactiae LMG 14609 fig|1080071.3.peg.332 Streptococcus orisasini fig|1139219.3.peg.2194 Enterococcus dispar ATCC 51266 fig|1834176.3.peg.811 Enterococcus sp. 3G1_DIV0629 fig|1622.15.peg.947 Lactobacillus murinus fig|565651.6.peg.1942 Enterococcus faecalis ARO1/DG fig|1473546.3.peg.703 Lysinibacillus sp. BF-4 fig|37734.13.peg.137 Enterococcus casseliflavus fig|492670.92.peg.623 Bacillus velezensis fig|1639.1907.peg.2641 Listeria monocytogenes fig|1123489.3.peg.170 Veillonella magna DSM 19857 fig|1280687.3.peg.1880 Butyrivibrio fibrisolvens YRB2005 fig|1262889.3.peg.680 Eubacterium sp. CAG:38 fig|1235800.3.peg.2226 Lachnospiraceae bacterium 10-1 fig|1897035.3.peg.445 Firmicutes bacterium GAG:552 39 19 fig|199.588.peg.774 Campylobacter concisus fig|1111133.4.peg.219 Peptoniphilus sp. BV3AC2 fig|936589.3.peg.875 Veillonella sp. AS16 WP_070600378.1 fig|1896998.3.peg.1750 Coprococcus sp. CAG:131- related_45_246 fig|41170.3.peg.3013 Exiguobacterium acetylicum fig|159620.44.peg.897 uncultured Clostridium sp. fig|1262843.3.peg.313 Clostridium sp. CAG:813 fig|1262834.3.peg.1287 Clostridium sp. CAG:715 fig|1256219.3.peg.760 Lactobacillus paracasei subsp. paracasei Lpp230 fig|115778.31.peg.1994 Leuconostoc gelidum subsp. gasicomitatum fig|29385.174.peg.531 Staphylococcus saprophyticus fig|1295.21.peg.75 Staphylococcus schleiferi fig|148814.13.peg.1360 Lactobacillus kunkeei fig|1282.1242.peg.673 Staphylococcus epidermidis fig|1581078.3.peg.1186 Staphylococcus sp. HMSC10C03 fig|1891097.3.peg.280 Macrococcus goetzii WP_080703103.1 fig|1214184.3.peg.1129 Streptococcus suis 22083 fig|1154771.3.peg.209 Streptococcus agalactiae FSL C1-487 fig|1415765.3.peg.1578 Streptococcus mitis 21/39 fig|1581074.3.peg.720 Granulicatella sp. HMSC31F03 fig|1349.233.peg.712 Streptococcus uberis fig|1946281.3.peg.392 Catabacter sp. UBA5893 fig|1328309.5.peg.1889 Lactobacillus plantarum IPLA88 fig|1214190.3.peg.2034 Streptococcus suis YS17 fig|29385.135.peg.2098 Staphylococcus saprophyticus fig|1715184.3.peg.1265 Aerococcus sp. HMSC035BO7 fig|1881068.3.peg.2940 Sphingomonas sp. OV641 fig|1522072.3.peg.3829 Sphingobium sp. bal fig|1802172.3.peg.237 Sphingopyxis sp. RIFCSPHIGHO2_12_FULL_65_19 fig|1128204.3.peg.2189 Bradyrhizobium elkanii CCBAU 43297 fig|1708715.5.peg.4517 Ensifer aridi fig|195105.3.peg.2062 Haematobacter massiliensis fig|1283312.3.peg.4182 Sphingomonas wittichii DC-6 fig|1120654.4.peg.406 Ensifer sp. LC499 fig|529.36.peg.3144 Ochrobactrum anthropi fig|1194716.3.peg.4774 Sinorhizobium meliloti AK75 fig|1660088.4.peg.2967 Agrobacterium sp. SCN 61-19 fig|1951259.3.peg.2515 Sphingomonadales bacterium UBA6174 fig|1912891.5.peg.2102 Sphingobium sp. fig|1670800.3.peg.1844 Mesorhizobium oceanicum fig|12032658.3.peg.157 Alphaproteobacteria bacterium WMHbin7 fig|1819565.5.peg.2208 Flavimaricola marinus fig|1245469.3.peg.1160 Bradyrhizobium oligotrophicum S58 fig|1615890.4.peg.173 Bradyrhizobium sp. LTSP849 fig|56454.3.peg.3464 Xanthomonas hortorum fig|40324.384.peg.1060 Stenotrophomonas maltophilia fig|1801972.3.peg.1832 Planctomycetes bacterium RBG_19FT_COMBO_48_8 fig|1978765.3.peg.3488 Nitrospira sp. ST-bin5 fig|2009322.3.peg.2770 Leptolyngbya ohadii IS1 fig|1325564.3.peg.3733 Nitrospira japonica fig|43662.9.peg.1688 Pseudoalteromonas piscicida fig|670.134.peg.4439 Vibrio parahaemolyticus fig|998520.3.peg.3325 Pseudoalteromonas agarivorans fig|1723759.3.peg.401 Pseudoalteromonas sp. P1-26 fig|672.133.peg.585 Vibrio vulnificus fig|1324960.19.peg.585 Aeromonas salmonicida subsp. pectinolytica 34mel fig|196024.16.peg.3965 Aeromonas dhakensis fig|654.27.peg.4266 Aeromonas veronii fig|1802253.3.peg.1045 Sulfurimonas sp. RIFCSPLOWO2_12_36_12 fig|636.16.peg.3905 Edwardsiella tarda fig|1124958.3.peg.5012 Salmonella enterica subsp. enterica serovar Muenster str. 0315 fig|573.10007.peg.225 Klebsiella pneumoniae fig|1946737.3.peg.4002 Leclercia sp. UBA1284 fig|1398203.3.peg.3712 Xenorhabdus bovienii str. kraussei Quebec fig|615.247.peg.2151 Serratia marcescens fig|52441.3.peg.3752 Nitrosomonas aestuarii fig|1951948.3.peg.242 Hyphomonadaceae bacterium UBA2389 fig|165186.29.peg.27 uncultured Ruminococcus sp. fig|2013842.3.peg.1881 Synergistetes bacterium HGW-Synergistetes-1 fig|411484.7.peg.436 Clostridium sp. SS2/1 fig|460384.4.peg.447 Enterocloster lavalensis fig|1761781.3.peg.2961 Clostridium sp. DSM 8431 fig|1451.25.peg.614 Paenibacillus amylolyticus fig|1776378.3.peg.2009 Paenibacillus phocaensis fig|1866315.3.peg.2122 Bacillus sp. N35-10-4 fig|1034836.4.peg.4077 Bacillus amyloliquefaciens XH7 fig|1397.14.peg.5097 Bacillus circulans fig|1497681.5.peg.772 Listeria newyorkensis fig|1053224.3.peg.4333 Bacillus cereus VD021 fig|1374.4.peg.2798 Pianococcus kocurii fig|458233.11.peg.419 Macrococcus caseolyticus JCSC5402 fig|417368.6.peg.944 Enterococcus thailandicus fig|1353.16.peg.736 Enterococcus gallinarum fig|1639.1307.peg.2578 Listeria monocytogenes fig|1649188.4.peg.450 Listeria goaensis fig|333990.5.peg.1279 Carnobacterium sp. AT7 fig|1121085.3.peg.4805 Bacillus aidingensis DSM 18341 fig|659243.6.peg.1163 Bacillus siamensis fig|1965645.3.peg.1428 Alistipes sp. An54 fig|1950664.3.peg.363 Bacteroidales bacterium UBA5918 fig|681398.3.peg.1596 Paludibacter jiangxiensis fig|1947481.3.peg.1596 Sphingobacterium sp. UBA1498 fig|1946424.3.peg.2345 Dysgonomonas sp. UBA4861 fig|188932.3.peg.968 Pedobacter cryoconitis fig|1505249.7.peg.1802 Arcobacter marinus fig|1802259.3.peg.374 Sulfurimonas sp. RIFOXYD12_FULL_33_39 fig|1872629.13.peg.663 Arcobacter sp. fig|497650.4.peg.949 Sulfurovum sp. enrichment culture clone C5 fig|1981711.3.peg.707 Pseudomonas sp. B8(2017) fig|287.926.peg.3808 Pseudomonas aeruginosa fig|157782.3.peg.183 Pseudomonas parafulva fig|1225174.5.peg.576 Pseudomonas mendocina S5.2 fig|237610.8.peg.4301 Pseudomonas psychrotolerans fig|1116369.3.peg.182 Hoeflea sp. 108 WP_080858354.1 fig|1679460.3.peg.2715 Marinibacterium profundimaris fig|1811547.3.peg.510 Maritimibacter sp. REDSEA-S28_B5 fig|93684.8.peg.518 Roseivivax halotolerans EMZ69714.1 Escherichia coli 174900 fig|103796.87.peg.3165 Pseudomonas syringae pv. actinidiae WP_078828851.1 Pantoea ananatis fig|2018067.3.peg.1734 Pseudomonas sp. FDAARGOS_380 fig|294.255.peg.5151 Pseudomonas fluorescens fig|287.4271.peg.5445 Pseudomonas aeruginosa fig|46677.3.peg.3237 Pseudomonas agarici fig|83964.10.peg.849 Pseudomonas coronafaciens pv. porri fig|1932113.4.peg.2793 Pseudomonas sp. PA1(2017) fig|1712677.3.peg.189 Pseudomonas sp. 2822-15 fig|1479235.3.peg.2741 Halomonas sp. HL-48 fig|227946.12.peg.3247 Xanthomonas translucens pv. poae fig|40324.220.peg.2801 Stenotrophomonas maltophilia fig|227946.13.peg.35 Xanthomonas translucens pv. poae fig|487909.15.peg.4212 Xanthomonas translucens pv. undulosa fig|40324.145.peg.2120 Stenotrophomonas maltophilia fig|1182783.3.peg.8 Xanthomonas campestris JX fig|1736581.3.peg.4144 Lysobacter sp. Root667 fig|470.4256.peg.2128 Acinetobacter baumannii fig|1804984.3.peg.4735 Burkholderia sp. OLGA172 fig|1882792.3.peg.5959 Burkholderia sp. CF145 fig|1458357.5.peg.7849 Caballeronia jiangsuensis fig|674703.3.peg.3992 Rhodoplanes sp. Z2-YC6860 fig|1230476.3.peg.595 Bradyrhizobium sp. DFCI-1 fig|1752222.3.peg.1730 Rhizobiales bacterium Ga0077525 fig|1948848.3.peg.320 Patescibacteria group bacterium UBA6220 fig|1860092.3.peg.3966 Alphaproteobacteria bacterium MedPE-SWcel fig|398.7.peg.3301 Rhizobium tropici fig|418630.3.peg.960 Rhodobacter megalophilus fig|56.40.peg.5712 Sorangium cellulosum fig|1660160.3.peg.2510 Acidobacteria bacterium SCN 69-37 fig|1661042.3.peg.2224 Pseudomonas sp. NBRC 111127 fig|1712678.3.peg.4198 Pseudomonas sp. 2822-17 fig|1736561.3.peg.128 Pseudomonas sp. Root562 fig|76760.8.peg.1730 Pseudomonas rhodesiae fig|1295133.4.peg.7170 Pseudomonas putida JCM 18798 fig|1718917.3.peg.3132 Pseudomonas sp. ICMP 460 fig|237306.3.peg.591 Pseudomonas syringae pv. persicae fig|1079060.3.peg.1479 Pseudomonas savastanoi pv. phaseolicola 1644R fig|1981714.3.peg.1068 Pseudomonas sp. B5(2017) fig|1419583.3.peg.4516 Pseudomonas mandelii PD30 fig|1718918.3.peg.4166 Pseudomonas sp. ICMP 561 fig|64988.7.peg.76 Alcanivorax jadensis fig|1961564.3.peg.685 Desulfovibrionaceae bacterium UBA5546 fig|2004648.3.peg.1747 Acinetobacter sp. WCHA39 fig|1080187.3.peg.399 Cupriavidus sp. UYPR2.512 fig|76114.8.peg.258 Aromatoleum aromaticum EbN1 fig|196367.9.peg.6286 Caballeronia sordidicola fig|1217418.3.peg.694 Cupriavidus sp. HPC(L) fig|1752216.3.peg.4007 Nitrosomonadales bacterium Ga0074132 fig|1249621.3.peg.3614 Cupriavidus sp. HMR-1 fig|179879.8.peg.6514 Burkholderia anthina fig|1246301.3.peg.4482 Variovorax paradoxus B4 WP_092746164.1 Acidovorax valerianellae fig|536.30.peg.857 Chromobacterium violaceum fig|1961112.3.peg.115 Planctomycetes bacterium UTPLA1 fig|44574.5.peg.4575 Nitrosomonas communis fig|265901.4.peg.190 Photobacterium sp. J15 fig|80852.21.peg.1289 Aliivibrio wodanis fig|1136159.3.peg.2534 Vibrio cyclitrophicus 1F111 fig|24.6.peg.4539 Shewanella putrefaciens fig|888433.3.peg.1974 Pseudoalteromonas sp. GutCa3 fig|196024.6.peg.3651 Aeromonas dhakensis fig|1352943.3.peg.5028 Vibrio harveyi E385 WP_088124663.1 Vibrio cholerae fig|29497.15.peg.1220 Vibrio splendidus fig|670.413.peg.1227 Vibrio parahaemolyticus fig|584.91.peg.3337 Proteus mirabilis fig|263819.5.peg.201 Yersinia aleksiciae fig|1656094.3.peg.1449 Alteromonas confluentis fig|634.5.peg.607 Yersinia bercovieri fig|630.85.peg.4080 Yersinia enterocolitica fig|1212491.3.peg.1847 Legionella fallonii LLAP-10 fig|1498499.3.peg.2812 Legionella norrlandica fig|1844092.4.peg.3143 Pseudomonas sp. 8 R 14 fig|1441629.3.peg.2119 Pseudomonas cichorii JBC1 WP_092369835.1 Pseudomonas seleniipraecipitans fig|477228.3.peg.2040 Pseudomonas stutzeri TS44 fig|317.249.peg.4299 Pseudomonas syringae fig|1597.16.peg.2055 Lactobacillus paracasei fig|1184720.6.peg.2708 Rhizobium anhuiense fig|1566263.3.peg.185 Rhizobium sp. NFR03 fig|1951216.3.peg.1622 Rhizobiales bacterium UBA1909 fig|1219052.3.peg.3572 Sphingomonas pruni NBRC 15498 fig|376620.8.peg.l22 Gluconobacter japonicus fig|1736587.3.peg.2638 Devosia sp. Root685 WP_011269850.1 Xanthomonas campestris fig|1195246.3.peg.467 Alishewanella agri BL06 fig|1931276.3.peg.1294 Haliangium sp. UPWRP 2 fig|1931204.4.peg.12 Confluentimicrobium sp. fig12052957.3.peg.3497 Pseudorhodobacter sp. MZDSW-24AT fig|1952825.3.peg.2772 Rhodobiaceae bacterium UBA4205 fig|1189622.3.peg.1716 Pseudomonas amygdali pv. tabaci str. 6605 fig|294.173.peg.2741 Pseudomonas fluorescens fig|294.122.peg.3307 Pseudomonas fluorescens fig|1198456.3.peg.4053 Pseudomonas guguanensis fig|1855380.3.peg.1951 Pseudomonas sp. Z003-0.4C(8344-21) fig|1144330.3.peg.3879 Pseudomonas sp. GM48 fig|86265.3.peg.2799 Pseudomonas thivervalensis fig|1881035.3.peg.3817 Mitsuaria sp. PDC51 fig|511.8.peg.774 Alcaligenes faecalis fig|1095552.3.peg.2955 Methylobacter luteus IMV-B-3098 fig|1690268.3.peg.1172 Acidovorax sp. SD340 fig|871652.3.peg.1451 Poseidonocella sedimentorum fig|1946868.3.peg.175 Methylophaga sp. UBA1490 fig|1924940.3.peg.1147 Mameliella sp. fig|1912891.7.peg.5370 Sphingobium sp. fig|1236503.3.peg.1539 Acetobacter persici JCM 25330 fig|1745182.3.peg.1942 Paracoccus sp. MKU1 fig|1112.5.peg.2875 Porphyrobacter neustonensis WP_051585410.1 Sphingomonas paucimobilis fig|1082931.4.peg.3584 Pelagibacterium halotolerans B2 fig|1907665.3.peg.5475 Agrobacterium sp. DSM 25558 fig|1841652.4.peg.3782 Agrobacterium sp. 13-626 fig|1736312.3.peg.3441 Rhizobium sp. Leaf262 fig|1768770.3.peg.4687 Caulobacter sp. CCH5-E12 fig|355591.9.peg.1867 Marinobacter vinifirmus fig|1869214.4.peg.1848 Rheinheimera sp. fig|1946470.3.peg.3546 Erythrobacter sp. UBA2510 fig|1860090.3.peg.231 Roseobacter sp. MedPE-SWde fig|12020902.8.peg.1814 Ponticaulis sp. fig|940286.3.peg.3612 Komagataeibacter oboediens 174Bp2 fig|1736380.3.peg.1842 Rhizobium sp. Leaf453 fig|665126.3.peg.2283 Prosthecomicrobium hirschii fig|2029410.3.peg.1956 Mesorhizobium sp. WSM4311 WP_003169203.1 Brevundimonas diminuta fig|1884373.3.peg.3317 Mesorhizobium sp. YR577 fig|989436.3.peg.3203 Pseudovibrio sp. Ad5 fig|1736359.3.peg.3976 Rhizobium sp. Leaf386 fig|104102.12.peg.3797 Acetobacter tropicalis fig|1500305.3.peg.4736 Rhizobium sp. OK665 fig|1842535.30.peg.6 Blastomonas sp. RAC04 fig|70775.16.peg.91 Pseudomonas plecoglossicida fig|287.4262.peg.2063 Pseudomonas aeruginosa WP_017702484.1 Pseudomonas syringae fig|1357292.3.peg.4700 Pseudomonas syringae pv. pisi str. PP1 fig|287.2436.peg.3554 Pseudomonas aeruginosa fig|76758.3.peg.4722 Pseudomonas orientalis fig|1904755.3.peg.3469 Pseudomonas sp. 43NM1 fig|47879.37.peg.693 Pseudomonas corrugata fig|1771311.3.peg.1935 Pseudomonas sp. ATCC PTA-122608 fig|2008975.3.peg.1976 Pseudomonas sp. Irchel 3E13 fig|1259798.3.peg.1121 Pseudomonas sp. LAMO17WK12:I2 fig|1736487.3.peg.2103 Noviherbaspirillum sp. Root189 fig|1706231.5.peg.1557 Janthinobacterium sp. CG23_2 fig|1804984.3.peg.4700 Burkholderia sp. OLGA172 fig|54067.3.peg.2925 Xylophilus ampelinus fig|40324.357.peg.1426 Stenotrophomonas maltophilia fig|1967657.4.peg.913 Salmonella enterica subsp. enterica serovar Telelkebir fig|573.14330.peg.816 Klebsiella pneumoniae fig|615.357.peg.16 Serratia marcescens fig|1122616.3.peg.231 Oceanospirillum beijerinckii DSM 7166 fig|314276.4.peg.1389 Idiomarina baltica OS145 fig|1038921.4.peg.2790 Pseudomonas chlororaphis subsp. aureofaciens 30-84 fig|292.72.peg.3280 Burkholderia cepacia fig|1899355.18.peg.947 Oceanospirillaceae bacterium fig|2015356.3.peg.5401 Burkholderia sp. AU33647 fig|206665.3.peg.1731 Desulfonauticus submarinus fig|1987165.3.peg.2564 Sphingobium sp. GW456-12-10-14-TSB1 fig|1283312.3.peg.2182 Sphingomonas wittichii DC-6 fig|1223566.3.peg.1810 Bradyrhizobium sp. CCGE-LA001 fig|76761.16.peg.744 Pseudomonas veronii PIY00499.1 Hydrogenophilales bacterium CG_4_10_14_3_um_filter_58_23 fig|305.94.peg.4778 Ralstonia solanacearum fig|1758178.5.peg.2546 Celeribacter ethanolicus fig|1354263.4.peg.2524 Hafnia paralvei ATCC 29927 fig|1125979.3.peg.1941 Rhizobium sp. PDO1-076 fig|1338032.3.peg.3393 Vibrio parahaemolyticus O1:K33 str. CDC_K4557 fig|1898112.54.peg.3344 Rhodospirillaceae bacterium fig|1432558.3.peg.4265 Klebsiella pneumoniae ISC21 fig|333962.3.peg.2767 Providencia heimbachae fig|60552.10.peg.2414 Burkholderia vietnamiensis WP_011808964.1 Verminephrobacter eiseniae fig|1844107.4.peg.2966 Pseudomonas sp. 58 R 12 fig|1952916.3.peg.906 Synergistaceae bacterium UBA5549 fig|1458817.8.peg.542 Shewanella halifaxensis HAW-EB4 fig|1674859.3.peg.1291 Spirochaetales bacterium Spiro_03 fig|1121434.3.peg.22 Halodesulfovibrio aestuarii DSM 10141 fig|1262899.3.peg.286 Fusobacterium sp. CAG:439 fig|57320.3.peg.1084 Pseudodesulfovibrio profundus fig|1736444.3.peg.3753 Acinetobacter sp. Root1280 fig|1310670.3.peg.2122 Acinetobacter sp. 907131 fig|505345.6.peg.150 Gallibacterium genomosp. 3 fig|670.887.peg.4391 Vibrio parahaemolyticus fig|196024.16.peg.2750 Aeromonas dhakensis fig|663.48.peg.1321 Vibrio alginolyticus fig|28141.133.peg.4717 Cronobacter sakazakii fig|1117315.3.peg.3 Pseudoalteromonas haloplanktis ATCC 14393 fig|1917164.4.peg.2739 Shewanella sp. UCD-KL21 fig|2006083.3.peg.3222 Photobacterium sp. CECT 9192 fig|584.227.peg.2146 Proteus mirabilis fig|1792834.4.peg.1793 Marinicella sediminis fig|1333513.3.peg.3775 Pseudoalteromonas haloplanktis TAE56 fig|1305826.3.peg.1246 Streptomyces sp. Ame12xC10 WP_048809063.1 Microbacterium ginsengisoli fig|1987376.3.peg.4246 Pseudonocardia sp. N23 fig|164115.3.peg.6832 Streptomyces niveiscabiei fig|285676.33.peg.4994 Micromonospora saelicesensis SFF52649.1 Streptomyces alni fig|1100822.3.peg.6408 Streptomyces sp. AmelKG-E11A WP_098467790.1 fig|1190417.3.peg.2916 Geodermatophilus telluris SDS16714.1 Agrococcus carbonis fig|692370.5.peg.1108 Altererythrobacter dongtanensis fig|1736370.3.peg.1383 Sphingomonas sp. Leaf412 fig|1759074.3.peg.2615 Sphingopyxis sp. HIX fig|1120928.3.peg.922 Acinetobacter tjernbergiae DSM 14971 = CIP 107465 fig|470.4268.peg.2032 Acinetobacter baumannii fig|1217627.3.peg.995 Acinetobacter baumannii NIPH 67 fig|28450.149.peg.5786 Burkholderia pseudomallei fig|2032650.3.peg.3543 Magnetococcales bacterium HCHbin5 fig|101571.169.peg.3909 Burkholderia ubonensis fig|396597.7.peg.2128 Burkholderia ambifaria MEX-5 fig|869212.3.peg.3514 Turneriella parva DSM 21527 fig|1196083.80.peg.1885 Snodgrassella alvi fig|1304886.3.peg.1257 Desulfotignum balticum DSM 7044 fig|555.16.peg.1362 Pectobacterium carotovorum subsp. carotovorum fig|1421338.3.peg.151 Enterobacter asburiae L1 fig|443144.3.peg.785 Geobacter sp. M21 fig|1265503.3.peg.1271 Colwellia piezophila ATCC BAA-637 fig|55601.100.peg.1601 Vibrio anguillarum fig|299766.9.peg.4890 Enterobacter hormaechei subsp. steigerwaltii fig|243231.5.peg.1360 Geobacter sulfurreducens PGA fig|1263083.3.peg.558 Klebsiella variicola CAG:634 fig|57706.9.peg.1106 Citrobacter braakii fig|1619244.3.peg.1323 Enterobacter bugandensis SHO56340.1 Vibrio quintilis fig|688.15.peg.906 Aliivibrio logei fig|663.75.peg.4800 Vibrio alginolyticus fig|1967612.3.peg.4508 Salmonella enterica subsp. houtenae serovar 50:z4, z23:- fig|82985.3.peg.3508 Pragia fontium fig|55207.5.peg.1840 Pectobacterium betavasculorum fig|582.25.peg.388 Morganella morganii fig|1006598.5.peg.80 Serratia plymuthica RVH1 fig|82977.3.peg.3268 Buttiauxella agrestis CRY53703.1 Yersinia intermedia fig|595494.3.peg.706 Tolumonas auensis DSM 9187 fig|1217694.3.peg.3300 Acinetobacter sp. CIP 64.2 fig|470.2679.peg.715 Acinetobacter baumannii fig|1879050.4.peg.2797 Acinetobacter wuhouensis fig|2004650.3.peg.1818 Acinetobacter chinensis fig|648.80.peg.1405 Aeromonas caviae fig|1217722.3.peg.1866 Pseudomonas sp. S13.1.2 fig|294.88.peg.4234 Pseudomonas fluorescens fig|629262.5.peg.1917 Pseudomonas syringae pv. japonica str. M301072 WP_053932309.1 Pseudomonas coronafaciens fig|1844101.3.peg.4702 Pseudomonas sp. 31 R 17 fig|380021.13.peg.6149 Pseudomonas protegens fig|287.3716.peg.2163 Pseudomonas aeruginosa fig|287.3208.peg.1464 Pseudomonas aeruginosa fig|1952221.3.peg.555 Methylococcaceae bacterium UBA2780 fig|1869214.3.peg.3542 Rheinheimera sp. fig|375286.7.peg.830 Janthinobacterium sp. Marseille fig|536.26.peg.4616 Chromobacterium violaceum fig|983548.3.peg.2977 Dokdonia sp. 4H-3-7-5 fig|1307480.5.peg.1780 Chryseobacterium vrystaatense fig|1262921.3.peg.2213 Prevotella sp. CAG:1185 fig|1965649.3.peg.4193 Butyricimonas sp. An62 fig|1951558.3.peg.3731 Chitinophagaceae bacterium UBA4411 fig|1950669.3.peg.2035 Bacteroidales bacterium UBA6192 fig|1869230.3.peg.3025 Chryseobacterium sp. CBo1 fig|1500294.3.peg.2814 Chryseobacterium sp. YR485 fig|1756149.11.peg.2545 Elizabethkingia bruuniana fig|1137281.3.peg.1436 Xanthomarina gelatinilytica fig|1964365.5.peg.2525 Sneathiella sp. fig|28450.428.peg.5049 Burkholderia pseudomallei fig|1628751.3.peg.813 Nostoc linckia z16 fig|60137.10.peg.1138 Sulfitobacter pontiacus fig|1580596.3.peg.2701 Phaeobacter piscinae fig|1041141.4.peg.4741 Rhizobium leguminosarum bv. viciae 128C53 WP_063290764.1 unclassified Pseudovibrio fig|1816219.4.peg.1873 Colwellia sp. PAMC 21821 fig|651.3.peg.13 Aeromonas media fig|134375.17.peg.3222 Achromobacter sp. WP_011296194.1 Cupriavidus pinatubonensis fig|1513890.4.peg.2322 Pseudomonas chlororaphis subsp. piscium fig|294.193.peg.980 Pseudomonas fluorescens fig|287.2516.peg.114 Pseudomonas aeruginosa fig|2006083.3.peg.3219 Photobacterium sp. CECT 9192 fig|1458817.8.peg.538 Shewanella halifaxensis HAW-EB4 fig|1073383.3.peg.1289 Aeromonas veronii AMC34 fig|190893.14.peg.2110 Vibrio coralliilyticus fig|663.144.peg.2659 Vibrio alginolyticus fig|55601.106.peg.926 Vibrio anguillarum fig|669.51.peg.5531 Vibrio harveyi fig|1250059.5.peg.3511 Tenacibaculum sp. MAR_2009_124 fig|906888.6.peg.2449 Nonlabens ulvanivorans WP_042276051.1 Nonlabens sediminis fig|1953167.3.peg.1254 Bacteroidetes bacterium UBA6221 fig|991.14.peg.629 Flavobacterium hydatis fig|1121890.3.peg.5 Flavobacterium frigidarium DSM 17623 fig|387094.4.peg.1115 Flavobacterium hercynium fig|1946558.3.peg.2413 Flavobacterium sp. UBA7665 fig|253.27.peg.2882 Chryseobacterium indologenes fig|1685010.5.peg.4376 Chryseobacterium glaciei fig|1500289.3.peg.4103 Chryseobacterium sp. OV705 fig|1500298.3.peg.2823 Chryseobacterium sp. YR561 fig|1797331.3.peg.2180 Bacteroidetes bacterium GWE2_29_8 fig|1947498.3.peg.1366 Sphingobacterium sp. UBA4616 WP_074239321.1 Chitinophaga niabensis fig|192149.7.peg.174 Muricauda sp. fig|718222.3.peg.4924 Bacillus cereus TIAC219 fig|1053210.3.peg.211 Bacillus cereus HuB4-10 fig|2026089.3.peg.6077 Paenibacillus sp. XY044 fig|1938610.3.peg.3678 Flavobacterium sp. LM5 fig|1947482.3.peg.632 Sphingobacterium sp. UBA1575 fig|1948844.3.peg.823 Patescibacteria group bacterium UBA6130 fig|986.7.peg.83 Flavobacterium johnsoniae fig|1950382.3.peg.461 Bacteroidales bacterium UBA1181 fig|1947145.3.peg.376 Prevotella sp. UBA3765 fig|1122989.3.peg.367 Prevotella oris DSM 18711 = JCM 12252 fig|1896974.3.peg.2001 Bacteroides sp. 43 108 fig|2014804.3.peg.4306 Lewinellaceae bacterium SD302 fig|1428.517.peg.2380 Bacillus thuringiensis fig|1428.574.peg.3351 Bacillus thuringiensis fig|1428.590.peg.5685 Bacillus thuringiensis fig|1720554.3.peg.188 Hungateiclostridium clariflavum DSM 19732 fig|1122203.4.peg.2283 Marinococcus halotolerans DSM 16375 fig|1462525.3.peg.3489 Thalassobacillus sp. TM-1 fig|1395513.3.peg.363 Sporolactobacillus laevolacticus DSM 442 fig|1262834.3.peg.1229 Clostridium sp. CAG:715 SCI87282.1 uncultured Roseburia sp. fig|1952116.3.peg.2349 Lachnospiraceae bacterium UBA6480 WP_069150959.1 Lachnospiraceae fig|1265.10.peg.3100 Ruminococcus flavefaciens fig|1120998.3.peg.2858 Anaerovorax odorimutans DSM 5092 WP_072702499.1 Butyrivibrio hungatei fig|1232453.3.peg.2795 Clostridiales bacterium VE202-21 fig|39485.11.peg.251 Lachnospira eligens WP_072832325.1 fig|1509.24.peg.2487 Clostridium sporogenes SCI88558.1 uncultured Clostridium sp. fig|1490.6.peg.2635 Paraclostridium bifermentans fig|1947399.3.peg.1322 Hungateiclostridiaceae bacterium UBA3548 fig|1953138.3.peg.796 Bacteroidetes bacterium UBA1312 fig|1950875.3.peg.364 Clostridiales bacterium UBA4139 fig|1396.1518.peg.4860 Bacillus cereus fig|1305675.3.peg.2174 Bacillus solimangrovi fig|1423.436.peg.4365 Bacillus subtilis fig|361277.6.peg.1539 Terribacillus saccharophilus fig|1392.356.peg.4724 Bacillus anthracis fig|1053189.3.peg.4219 Bacillus cereus BAG5X1-1 WP_079442297.1 Clostridium chromiireducens fig|1953262.3.peg.783 Candidatus Omnitrophica bacterium UBA1562 fig|1797955.3.peg.2304 Elusimicrobia bacterium RIFOXYA12_FULL_51_18 fig|1953111.3.peg.2436 Acidobacteria bacterium UBA7540 WP_099010551.1 Escherichia coli Retron- Eco1 (Ec86) fig|1005565.3.peg.1153 Escherichia coli 3006 fig|158822.8.peg.1905 Cedecea neteri fig|1444060.3.peg.4830 Escherichia coli 4-203-08_S1_C1 fig|29484.22.peg.4571 Yersinia frederiksenii fig|529823.3.peg.332 Cellvibrio sp. OA-2007 fig|48296.218.peg.3142 Acinetobacter pittii fig|550.518.peg.3445 Enterobacter cloacae fig|204773.6.peg.4 Herminiimonas arsenicoxydans fig|670.190.peg.4348 Vibrio parahaemolyticus fig|44577.7.peg.209 Nitrosomonas ureae fig|1125747.3.peg.1 Paraglaciecola agarilytica NO2 fig|1338034.3.peg.2437 Vibrio parahaemolyticus O1:Kuk str. FDA_R31 fig|1952844.3.peg.2619 Rhodocyclaceae bacterium UBA5533 fig|1288788.3.peg.2384 Vibrio parahaemolyticus 3631 fig|644.31.peg.975 Aeromonas hydrophila fig|498292.3.peg.28 Flavobacterium swingsii fig|1948088.3.peg.4515 Firmicutes bacterium UBA6132 fig|1408433.3.peg.3094 Crocinitomix catalasitica ATCC 23190 WP_074236572.1 Chryseobacterium zeae fig|1127353.3.peg.1738 Salmonella enterica subsp. enterica serovar Newport str. #11-4 fig|1881110.4.peg.120 Pantoea sesami fig|34038.6.peg.27 Rahnella aquatilis fig|630.95.peg.4256 Yersinia enterocolitica fig|149387.11.peg.1139 Salmonella enterica subsp. enterica serovar Brandenburg fig|1343738.3.peg.2232 Vibrio cholerae 2012EL-1759 Retron- Vch3 (Vc137) fig|1423.175.peg.4339 Bacillus subtilis fig|2021695.3.peg.3399 Bacillus sp. 7894-2 fig|189426.10.peg.597 Paenibacillus odorifer fig|2020949.3.peg.856 Romboutsia weinsteinii fig|1243664.3.peg.1004 Bacillus massiliogorillae fig|1855345.3.peg.2971 Bacillus sp. RRD69 fig|1946358.3.peg.2514 Clostridium sp. UBA4108 fig|1520.90.peg.2502 Clostridium beijerinckii fig|79672.3.peg.288 Bacillus thuringiensis serovar medellin fig|189426.19.peg.3973 Paenibacillus odorifer fig|1497.3.peg.4049 Clostridium formicaceticum fig|169760.4.peg.4269 Paenibacillus stellifer WP_073588670.1 Anaerocolumna xylanovorans fig|1950815.3.peg.1585 Clostridiales bacterium UBA1341 fig|1897004.3.peg.2166 Eubacterium sp. 45_250 fig|1946293.3.peg.290 Catabacter sp. UBA7571 fig|1796620.3.peg.3489 Acutalibacter muris fig|76857.53.peg.2245 Fusobacterium nucleatum subsp. polymorphum fig|2013784.3.peg.1260 Firmicutes bacterium HGW- Firmicutes-3 fig|79884.3.peg.1106 Bacillus pseudalcaliphilus fig|135461.47.peg.1552 Bacillus subtilis subsp. subtilis WP_016122013.1 Bacillus cereus group fig|1499688.3.peg.3214 Bacillus sp. LF1 fig|1318.8.peg.1495 Streptococcus parasanguinis fig|1381091.3.peg.1173 Streptococcus equi subsp. zooepidemicus SzAM60 fig|1409369.3.peg.815 Staphylococcus aureus AMMC6050 fig|1497681.5.peg.3014 Listeria newyorkensis fig|1095727.3.peg.409 Streptococcus sp. SK643 fig|1318.12.peg.313 Streptococcus parasanguinis fig|1681184.3.peg.4899 Lysinibacillus sp. ZYM-1 fig|561879.29.peg.3569 Bacillus safensis fig|1884359.3.peg.2978 Psychrobacillus sp. OK028 fig|29367.3.peg.1112 Clostridium puniceum fig|1225345.3.peg.1103 Clostridium chromiireducens fig|1345695.10.peg.2438 Clostridium saccharobutylicum DSM 13864 fig|119641.3.peg.784 Clostridium uliginosum fig|1761781.3.peg.88 Clostridium sp. DSM 8431 fig|1946346.3.peg.2999 Clostridium sp. UBA1056 fig|1492.48.peg.3952 Clostridium butyricum fig|1121302.3.peg.4511 Clostridium cavendishii DSM 21758 fig|398512.4.peg.5301 Pseudobacteroides cellulosolvens ATCC 35603 = DSM 2933 fig|1946357.3.peg.500 Clostridium sp. UBA3947 fig|642492.3.peg.3338 Cellulosilyticum lentocellum DSM 5427 fig|1946690.3.peg.1553 Lachnoclostridium sp. UBA3320 fig|397290.3.peg.150 Lachnospiraceae bacterium A2 fig|97138.3.peg.1713 Clostridium sp. ASF356 fig|1965545.3.peg.499 Tyzzerella sp. An114 fig|1047063.3.peg.240 WS1 bacterium JGI 0000059-K21 fig|1192034.3.peg.1508 Chondromyces apiculatus DSM 436 AAA88323.1 Myxococcus xanthus Retron- Mxa2 (Mx65) fig|33.8.peg.8196 Myxococcus fulvus fig|215803.3.peg.1485 Enhygromyxa salina fig|1406225.3.peg.2150 Archangium violaceum Cb vi76 fig|1952931.3.peg.5137 Verrucomicrobia subdivision 3 bacterium UBA6082 fig|1972460.3.peg.279 Anaerolineaceae bacterium 4572_78 fig|1950201.3.peg.3297 Anaerolineales bacterium UBA2796 WP_015247705.1 fig|1799658.3.peg.2777 Planctomycetaceae bacterium SCGC AG-212-F19 fig|214688.26.peg.6659 Gemmata obscuriglobus UQM 2246 fig|12023130.3.peg.4250 Rhodopirellula sp. MGV fig|52.7.peg.9046 Chondromyces crocatus fig|448385.16.peg.2914 Sorangium cellulosum So ce56 fig|888845.4.peg.14202 Minicystis rosea fig|1752210.3.peg.1275 Deltaproteobacteria bacterium Ga0077539 fig|1391654.3.peg.3562 Labilithrix luteola fig|1752218.3.peg.3670 Planctomycetaceae bacterium Ga0077529 WP_006981058.1 Chthoniobacter flavus fig|1952939.3.peg.2584 Verrucomicrobiaceae bacterium UBA1938 fig|2024858.3.peg.3711 Sandaracinus sp. WP_009096166.1 Rhodopirellula sp. SWK7 fig|595453.3.peg.1506 Rhodopirellula sp. SM50 fig|1263868.3.peg.4100 Rhodopirellula europaea SH398 fig|167547.3.peg.303 Prochlorococcus marinus str. MIT 9311 fig|1499501.3.peg.459 Prochlorococcus sp. SS52 fig|1905359.3.peg.4335 marine bacterium AO1-C WP_002700020.1 Microscilla marina fig|1913989.145.peg.263 Gammaproteobacteria bacterium WP_073154989.1 Seinonella peptonophila fig|46223.3.peg.3648 Thermoflavimicrobium dichotomicum fig|1329796.3.peg.1947 Risungbinella massiliensis fig|1123252.3.peg.3225 Shimazuella kribbensis DSM 45090 fig|714067.3.peg.3719 Kroppenstedtia eburnea fig|1341151.3.peg.628 Laceyella sacchari 1-1 fig|2026763.3.peg.3089 Myxococcales bacterium fig|1797895.3.peg.2518 Deltaproteobacteria bacterium RIFOXYA12_FULL_58_15 fig|373672.4.peg.4082 Chryseobacterium gambrini fig|1416778.5.peg.4374 Chryseobacterium arachidis fig|1603293.4.peg.829 Flavobacterium sp. 316 CCB70859.1 Flavobacterium branchiophilum FL-15 fig|1986952.3.peg.951 Sphingobacteriaceae bacterium GW460-11-11-14-LB5 fig|1476464.3.peg.4304 Pedobacter xixiisoli fig|1761785.3.peg.3112 Flavobacterium sp. ov086 fig|1664068.3.peg.3690 bacterium 336/3 fig|880071.3.peg.3500 Bernardetia litoralis DSM 6794 fig|1121902.3.peg.2906 Eisenibacter elegans DSM 3317 fig|1509483.4.peg.1923 Flectobacillus sp. BAB-3569 fig|1166018.3.peg.5312 Fibrella aestuarina BUZ 2 fig|634771.3.peg.967 Chitinophaga eiseniae fig|29529.3.peg.396 Chitinophaga arvensicola fig|1891659.3.peg.6032 Chitinophaga sp. CB10 fig|2033437.3.peg.3442 Chitinophaga sp. MD30 fig|1004.4.peg.1677 Chitinophaga sancti fig|1881041.3.peg.3698 Chitinophaga sp. YR627 fig|1123078.3.peg.2010 Runella zeae DSM 19591 fig|1354355.3.peg.1846 Niastella yeongjuensis fig|1951546.3.peg.1574 Chitinophagaceae bacterium UBA1946 fig|1812911.3.peg.633 Flavihumibacter sp. CACIAM 22H1 fig|477680.4.peg.4668 Filimonas lacunae fig|221126.7.peg.3781 Algibacter lectus fig|342954.4.peg.734 Lacinutrix algicola fig|1871037.5.peg.2180 Flavobacteriaceae bacterium fig|669041.3.peg.843 Tenacibaculum dicentrarchi WP_074538568.1 Cellulophaga baltica fig|1248440.3.peg.1511 Polaribacter franzmannii ATCC 700399 fig|1121007.3.peg.1574 Aquimarina muelleri DSM 19832 fig|688867.3.peg.2332 Ohtaekwangia koreensis fig|926565.3.peg.717 Sporocytophaga myxococcoides DSM 11118 fig|1257021.3.peg.5265 Flammeovirgaceae bacterium 311 fig|12044937.5.peg.2350 candidate division KSB3 bacterium fig|1499966.3.peg.180 Candidatus Moduliflexus flocculans fig|1948269.3.peg.1254 Verrucomicrobia bacterium UBA6053 fig|694433.3.peg.3103 Saprospira grandis DSM 2844 fig|2008677.3.peg.3337 Mitsuaria noduli fig|946333.3.peg.3570 Rhizobacter gummiphilus fig|1736433.3.peg.5559 Rhizobacter sp. Root1221 fig|1500265.3.peg.5760 Methylibium sp. YR605 fig|1121349.4.peg.2836 Comamonas composti DSM 21721 fig|1082851.3.peg.91 Comamonas serinivorans fig|1121480.5.peg.5468 Pseudoduganella violaceinigra DSM 15887 fig|2045208.3.peg.1247 Massilia violaceinigra fig|1736455.3.peg.3692 Massilia sp. Root133 fig|34073.25.peg.8569 Variovorax paradoxus fig|1884311.3.peg.7121 Variovorax sp. OK202 fig|1123487.3.peg.1763 Uliginosibacterium gangwonense DSM 18521 fig|2029111.3.peg.3032 Comamonadaceae bacterium NML120219 fig|1977087.20.peg.1226 Proteobacteria bacterium fig|754436.4.peg.4454 Photobacterium aphoticum fig|265726.7.peg.1038 Photobacterium halotolerans fig|1121867.3.peg.59 Enterovibrio calviensis DSM 14347 fig|1238431.3.peg.2655 Vibrio nigripulchritudo BLFn1 fig|1384589.3.peg.2721 [ Erwinia ] teleogrylli fig|1261127.3.peg.2947 Citrobacter amalonaticus Y19 fig|349521.8.peg.3588 Hahella chejuensis KCTC 2396 fig|1525918.3.peg.1501 Thiothrix caldifontis fig|1737490.4.peg.4974 Agarilytica rhodophyticola fig|251229.3.peg.427 Chroococcidiopsis thermalis PCC 7203 fig|1245923.3.peg.9587 Scytonema millei VB511283 fig|1503470.5.peg.10896 cyanobacterium TDX16 fig|2005460.3.peg.1118 Chondrocystis sp. NIES-4102 fig|179408.3.peg.4679 Oscillatoria nigro-viridis PCC 7112 fig|1612423.3.peg.5384 Nostoc linckia z1 fig|63737.11.peg.472 Nostoc punctiforme PCC 73102 fig|224013.5.peg.7163 Nostoc piscinale CENA21 fig|1932621.3.peg.7363 Nostoc sp. T09 fig|373994.3.peg.3383 Rivularia sp. PCC 7116 fig|2005463.3.peg.257 Calothrix sp. NIES-4105 fig|2005459.3.peg.7019 Tolypothrix sp. NIES-4075 fig|184925.3.peg.2602 Chlorogloeopsis fritschii PCC 9212 fig|454136.5.peg.3127 Phormidium ambiguum IAM M-71 fig1203124.6.peg.2732 Trichodesmium erythraeum IMS101 fig|2040638.3.peg.3067 Tychonema bourrellyi FEM_GT703 fig|1880991.4.peg.2927 Oscillatoriales cyanobacterium USR001 fig|1173028.3.peg.7115 Oscillatoria sp. PCC 10802 fig|568701.4.peg.2073 Moorea bouillonii PNG fig|927677.3.peg.4187 Synechocystis sp. PCC 7509 fig|179408.3.peg.6267 Oscillatoria nigro-viridis PCC 7112 fig|1710894.3.peg.2079 Aphanizomenon flos-aquae LD13 fig|1947888.3.peg.4484 Cyanobacteria bacterium UBA6047 fig|1705388.3.peg.1178 Planktothricoides sp. SR001 fig|454136.5.peg.4162 Phormidium ambiguum IAM M-71 fig|2005458.3.peg.378 Nostoc sp. NIES-4103 fig|1947874.3.peg.4590 Cyanobacteria bacterium UBA1583 fig|1781255.3.peg.802 Desertifilum sp. IPPAS B-1220 fig|1128427.4.peg.2346 filamentous cyanobacterium ESFC-1 fig|1946321.3.peg.3928 Chloracidobacterium sp. UBA7656 fig|118173.3.peg.1030 Pseudanabaena sp. PCC 6802 fig|1922337.4.peg.4802 Leptolyngbya sp. ‘hensonii’ fig|927668.3.peg.2766 Pseudanabaena biceps PCC 7429 fig|1173020.3.peg.6191 Chamaesiphon minutus PCC 6605 fig|329726.14.peg.4440 Acaryochloris marina MBIC11017 fig|215803.3.peg.1649 Enhygromyxa salina fig|1920190.3.peg.9548 Archangium sp. Cb G35 fig|1961464.3.peg.5181 Myxococcales bacterium UBA2376 fig|765913.3.peg.336 Thiorhodococcus drewsii AZ1 fig|1396141.3.peg.2891 Haloferula sp. BvORR071 fig|1961463.3.peg.5253 Myxococcales bacterium UBA1671 AAL40743.1 Nannocystis exedens Retron- Nex2 (Ne144) fig|54.3.peg.4123 Nannocystis exedens fig|53367.3.peg.3417 Asanoa ferruginea fig460265.11.peg.3882 Methylobacterium nodulans ORS 2060 fig|298794.3.peg.462 Methylobacterium variabile fig|190148.4.peg.3492 Bradyrhizobium paxllaeri fig|1075417.3.peg.445 Catalinimonas alkaloidigena fig|1429438.4.peg.7505 Candidatus Entotheonella factor fig|1977087.12.peg.1756 Proteobacteria bacterium fig|92487.3.peg.3972 Thiothrix eikelboomii fig|1977087.20.peg.1473 Proteobacteria bacterium fig|1123400.3.peg.3276 Thiofilum flexile DSM 14609 fig|34062.8.peg.73 Moraxella osloensis fig|1699623.3.peg.1502 Psychrobacter sp. P11G3 fig|1123509.3.peg.848 Zooshikella ganghwensis DSM 15267 fig|2026735.3.peg.2222 Deltaproteobacteria bacterium fig|1977087.12.peg.2982 Proteobacteria bacterium fig|2026763.4.peg.1195 Myxococcales bacterium fig|1977087.12.peg.510 Proteobacteria bacterium fig|1123508.3.peg.7252 Zavarzinella formosa DSM 19928 fig|214688.26.peg.3091 Gemmata obscuriglobus UQM 2246 fig|1908690.5.peg.1204 Fimbriiglobus ruber fig|1805126.3.peg.4431 Deltaproteobacteria bacterium CG2_30_63_29 fig|1882752.4.peg.1962 Singulisphaera sp. GP187 fig|1636152.3.peg.5364 Planctomyces sp. SH-PL62 APR75442.1 Minicystis rosea fig|54.3.peg.8798 Nannocystis exedens fig|980254.4.peg.4083 Roseimaritima ulvae fig|1856297.3.peg.3627 Gammaproteobacteria bacterium 45_16_T64 fig|1219077.3.peg.1945 Vibrio azureus NBRC 104587 fig|1334629.3.peg.167 Myxococcus fulvus 124B02 AAA25405.1 Myxococcus xanthus Retron- Mxa1 (Mx162) fig|378806.16.peg.4444 Stigmatella aurantiaca DW4/3-1 WP_002615305.1 Stigmatella aurantiaca Retron- Sau1 (Sa163) fig|48.3.peg.757 Archangium gephyra fig|448385.16.peg.2083 Sorangium cellulosum So ce56 fig|52.7.peg.5100 Chondromyces crocatus fig|1752210.3.peg.5621 Deltaproteobacteria bacterium Ga0077539 fig|2024858.3.peg.6345 Sandaracinus sp. WP_012826728.1 Haliangium ochraceum fig|927083.3.peg.3408 Sandaracinus amylolyticus WP_006977315.1 Plesiocystis pacifica fig|1400863.5.peg.627 Candidatus Competibacter denitrificans Run_A_D11 fig|1961463.3.peg.4303 Myxococcales bacterium UBA1671 fig|1898731.3.peg.3099 Curtobacterium sp. MCBA15_001 fig|1279028.3.peg.3374 Curtobacterium sp. 314Chir4.1 fig|1898733.3.peg.2056 Curtobacterium sp. MCBA15_004 fig|1795630.3.peg.3476 Frondihabitans sp. PAMC 28766 fig|2033654.3.peg.3461 Curtobacterium sp. ‘Ferrero’ fig|1736329.5.peg.1436 Frondihabitans sp. Leaf304 fig|1736292.3.peg.1083 Rathayibacter sp. Leaf185 fig|1736327.3.peg.206 Rathayibacter sp. Leaf296 fig|1736311.3.peg.3668 Curtobacterium sp. Leaf261 fig|1736308.3.peg.2333 Frigoribacterium sp. Leaf254 fig|656366.8.peg.2905 Arthrobacter alpinus fig|494023.3.peg.138 Paeniglutamicibacter antarcticus ASN40093.1 Arthrobacter sp. 7749 fig|1494608.3.peg.465 Arthrobacter sp. PAMC 25486 fig|656366.4.peg.2620 Arthrobacter alpinus fig|656366.3.peg.1944 Arthrobacter alpinus fig|1132441.3.peg.1888 Arthrobacter sp. 35W fig|1704044.3.peg.520 Arthrobacter sp. ERGS1:01 fig|1496689.3.peg.681 Arthrobacter sp. L77 fig|1681197.3.peg.149 Arthrobacter sp. RIT-PI-e fig|37921.12.peg.1481 Arthrobacter agilis fig|1736303.3.peg.982 Arthrobacter sp. Leaf234 fig|1312978.3.peg.1472 Arthrobacter sp. H41 fig|1348338.3.peg.1472 Leifsonia rubra CMS 76R fig|1452536.3.peg.1955 Microbacterium sp. Cr-K20 fig|1736525.3.peg.446 Leifsonia sp. Root4 fig|1529318.3.peg.434 Cryobacterium sp. MLB-32 fig|1267973.3.peg.3479 Arthrobacter sp. H5 fig|150121.3.peg.1900 Agreia pratensis fig|123316.3.peg.955 Agreia sp. VKM Ac-2052 fig|1052260.3.peg.3617 Klenkia soli fig|1566299.3.peg.3962 Klenkia marina fig|1736356.3.peg.3150 Modestobacter sp. Leaf380 fig|1736354.3.peg.1787 Geodermatophilus sp. Leaf369 fig|479431.6.peg.3115 Nakamurella multipartita DSM 44233 fig|1090615.3.peg.2397 Nakamurella panacisegetis fig|1306174.4.peg.4778 Kineosporia aurantiaca JCM 3230 fig|546871.3.peg.1120 Friedmanniella luteola fig|630515.4.peg.525 Microlunatus soli fig|546874.3.peg.1181 Friedmanniella sagamiharensis BAK35674.1 Microlunatus phosphovorus NM-1 fig|1380390.4.peg.72 Solirubrobacterales bacterium URHD0059 fig|1283299.3.peg.2784 Conexibacter woesei Iso977N fig|929712.3.peg.3165 Patulibacter minatonensis DSM 18081 fig|1123262.3.peg.3125 Solirubrobacter soli DSM 22325 fig|1861.4.peg.5240 Geodermatophilus obscuras fig|1137993.4.peg.1318 Geodermatophilus africanus fig|1070870.3.peg.778 Geodermatophilus nigrescens fig|1190417.3.peg.1785 Geodermatophilus telluris fig|477641.3.peg.1023 Modestobacter marinus fig|1798228.3.peg.3756 Blastococcus sp. DSM 46838 WP_091929708.1 Blastococcus sp. DSM 46786 SHH20361.1 Jatrophihabitans endophyticus fig|1844.3.peg.1151 Nocardioides luteus fig|748909.6.peg.1418 Nocardioides alpinus fig1402596.3.peg.987 Nocardioides exalbidus fig|1736322.3.peg.1963 Nocardioides sp. Leaf285 fig|1445613.3.peg.3490 Sciscionella sp. SE31 fig|543632.4.peg.9742 Actinoplanes subtropicus fig|1036182.3.peg.2958 Actinoplanes atraurantiacus fig|1246995.3.peg.737 Actinoplanes friuliensis DSM 7358 fig|56427.3.peg.3052 Couchioplanes caeruleus subsp. caeruleus fig|1710355.3.peg.2225 Actinoplanes sp. TFC3 fig|649831.3.peg.2352 Actinoplanes sp. N902-109 fig|35754.4.peg.6321 Dactylosporangium aurantiacum fig|1881.4.peg.2703 Micromonospora viridifaciens fig|47863.3.peg.3975 Micromonospora globosa fig|285665.4.peg.2050 Micromonospora coriariae fig|1192034.3.peg.4668 Chondromyces apiculatus DSM 436 fig|1198133.3.peg.2442 Myxococcus xanthus DZ2 fig|33.8.peg.521 Myxococcus fulvus fig|394193.3.peg.7794 Amycolatopsis saalfeldensis fig|369932.4.peg.5621 Amycolatopsis niigatensis fig|1238180.3.peg.5340 Amycolatopsis azurea DSM 43854 fig|589385.3.peg.7940 Amycolatopsis xylanica fig|1068980.3.peg.1527 Amycolatopsis nigrescens CSC17Ta-90 fig|1854586.3.peg.2100 Amycolatopsis antarctica fig|587909.3.peg.3086 Yuhushiella deserti fig|2030.3.peg.3051 Kibdelosporangium aridum fig|1382595.4.peg.3164 Saccharopolyspora erythraea D WP_013675061.1 Pseudonocardia dioxanivorans fig|1660131.3.peg.2805 Pseudonocardia sp. SCN 72-86 fig|366584.3.peg.4349 Pseudonocardia oroxyli fig|1885031.4.peg.5241 Pseudonocardia sp. Ae331 Ps2 fig|1690815.5.peg.5350 Pseudonocardia sp. HH130630-07 fig|1123023.3.peg.3229 Pseudonocardia acaciae DSM 45401 fig|1449976.3.peg.8114 Kutzneria albida DSM 43870 WP_007238159.1 fig|1220583.3.peg.1582 Gordonia aichiensis NBRC 108223 GAB07179.1 Gordonia amarae NBRC 15530 fig|1223545.3.peg.704 Gordonia soli NBRC 108243 fig|1223540.3.peg.3237 Gordonia desulfuricans NBRC 100010 fig|1112204.3.peg.4913 Gordonia polyisoprenivorans VH2 AFR49048.1 Gordonia sp. KTR9 fig|402289.3.peg.1558 Rhodococcus sp. HA99 fig|1077144.3.peg.224 Dietzia alimentaria 72 fig|1344003.3.peg.1864 Williamsia sterculiae fig|1463823.3.peg.3407 Microbispora sp. NRRL B-24597 fig|1903117.3.peg.1566 Williamsia sp. 1138 fig|1603258.4.peg.1828 Williamsia herbipolensis fig|644548.3.peg.652 Gordonia neofelifaecis NRRL B-59395 fig|1136941.3.peg.1310 Gordonia phthalatica fig|1223542.3.peg.3439 Gordonia malaquae NBRC 108250 fig|47312.10.peg.4232 Tsukamurella pulmonis fig|57704.14.peg.421 Tsukamurella tyrosinosolvens fig|521096.6.peg.2443 Tsukamurella paurometabola DSM 20162 fig|1123241.3.peg.3642 Nakamurella lactea DSM 19367 fig|1210073.4.peg.1031 Nocardia salmonicida NBRC 100378 fig|1206740.4.peg.4617 Nocardia thailandica NBRC 100428 fig|1210064.4.peg.2434 Nocardia altamirensis NBRC 108246 fig|1123258.3.peg.1651 Smaragdicoccus niigatensis DSM 44881 = NBRC 103563 fig|1443888.3.peg.2891 Rhodococcus fascians 02-815 fig|1517936.4.peg.882 Rhodococcus sp. CUA-806 fig1398843.6.peg.4214 Rhodococcus kyotonensis fig|1813677.3.peg.4031 Rhodococcus sp. EPR-157 WP_008711873.1 fig|1381122.3.peg.6103 Rhodococcus erythropolis DN1 fig|1736210.3.peg.2766 Rhodococcus sp. Leaf7 fig|1736300.3.peg.1279 Rhodococcus sp. Leaf225 fig|1219012.3.peg.1705 Rhodococcus corynebacterioides NBRC 14404 fig|1219023.3.peg.2791 Rhodococcus rhodnii NBRC 100604 21339 fig|616997.3.peg.2548 Hoyosella altamirensis 21340 fig|1303689.4.peg.2934 Rhodococcus koreensis JCM 10743 = 21341 NBRC 100607 ^(a)Accession in Patric database (https://www.patricbrc.org/) or NCBI (https://www.ncbi.nlm.nih.gov/protein/)

Table X provides non-limiting examples of 1928 retron reverse transcriptases (AA sequences associated with each NCBI Accession No. assigned to sequence identifiers SEQ ID NO: 19413-21341) that may be modified in accordance with the herein methods to obtain a recombinant retron reverse transcriptase for use in the compositions, systems, and methods described herein. These retron sequences were reported in Mestre et al., “Systematic Prediction of Genes Functionally Associated with Bacterial Retrons and Classification of The Encoded Tripartite Systems,” Nucleic Acids Research, Volume 48, Issue 22, 16 Dec. 2020, Pages 12632-12647, the contents of which are incorporated herein by reference. In some embodiments, the Table X retrons are meant to be excluded from the scope of the claimed subject matter.

Thus, in various aspects, the present disclosure describes making recombinant retrons and retron-based genome editing sy tables summarize the amino acid and nucleotide sequences disclosed herein, each being ascribed an individual sequence identifier (i.e., SEQ ID NO). In some instances, the sequences are disclosed in the body of the Specification. In other instances, the sequences are disclosed in the Sequence Listing, which forms a part of the instant Specification.

The following is a sequence key to summarize and identify the enclosed sequences. The sequence key accompanies the visual summary provided in FIGS. 28 and 29 .

Table A and Sub-Tables A1-A45: Novel Retron Reverse Transcriptase Sequences Seq ID Nos RT Amino RT Nucleic Functional Table Acid Sequence Acid Sequence Types A1 3980-4178 11231-11429 I-A A2 4671-4825 11922-12075 I-B1 A3 4980-5143 12229-12392 I-B2 A4 367-368, 427-441, 7624-7625, 7684- I-C 494-521, 526-527, 7698, 7751-7778, 536, 626, 649, 7783-7784, 7793, 660-668, 675, 679, 7883, 7906, 7917- 687-692, 695, 697, 7925, 7932, 7936, 703, 716, 721-722, 7944-7949, 7952, 751-763, 767, 770- 7954, 7960, 7973, 1411, 1456-1462 7978-7979, 8008- 8020, 8024, 8027- 8667, 8712-8718 A5 1529-1569 8784-8823 I-D A6 6697-6701 13943-13947 II A7 4179-4670 11430-11921 II-A1 A8 4884-4909 12134-12159 II-A1 other A9 6919-6972 14163-14215 II-A2 A10 2786-2866, 10039-10119, II-A3 2887-2938 10140-10191 A11 4826-4863 12076-12113 II-A4 A12 4864-4875 12114-12125 II-A4 fused A13 6974-7002 14217-14244 II-A5 A14 2598-2600, 9851-9853, III 2759-2785 10012-10038 A15 2445-2582 9699-9836 III-A1 A16 1983-2158 9237-9412 III-A2 A17 1612-1982 8866-9236 III-A3 A18 2601-2678 9854-9931 III-A4 A19 2679-2758 9932-10011 III-A5 A20 3442-3603 10694-10855 IV A21 3604-3708 10856-10959 V A22 2939-3441, 10192-10693, VI 3709-3979, 10960-11230, 5177-5192 12426-12441 A23 7003-7033 14245-14275 VII-A1 A24 7054-7133 14296-14374 VII-A2 A25 7034-7049 14276-14291 VIII A26 6835-6918 14079-14162 IX A27 6823-6834 14068-14078 X A28 298-366, 369-373, 7555-7623, 2626- XI 442-493, 522-525, 7630, 7699-7750, 528-535, 537, 551- 7785-7792, 7794, 554, 557, 560-625, 7808-7811, 7814, 672-674, 680-681, 7817-7882, 7929- 684-686, 696, 698, 7931, 7937-7938, 702, 723-742, 764- 7941-7943, 7953, 766, 1412-1450, 7955, 7959, 7980- 1452-1453, 1463- 7999, 8021-8023, 1466, 1571-1577 8668-8706, 8708- 8709, 8719-8722, 8825-8831 A29 374-426, 539-550, 7631-7683, 7796- XII 555-556, 558-559, 7807, 7812-7813, 671, 682-683, 743, 7815-7816, 7928, 745-750 7939-7940, 800, 8002-8007 A30 5942-6665 13189-13911 XIII A31 1-297, 715, 1580- 7258-7554, 7972, XIV 1603 8834-8857 A32 705-714 7962-7971 XV A33 6681-6694 13927-13940 XVI A34 6788-6803 14033-14048 XVII A35 1469-1526, 5147- 8725-8781, 12396- CRISPR- 5151 12400 associated A36 2159-2428 9413-9682 Ec107-like A37 646-648 7903-7905 RT-atpase A38 2592-2595 9846-9849 RT-DUF4116 A39 676-678, 717-720 7933-7935, 7974- RT-HTH 7977 A40 538, 669, 704, 7795, 7926, 7961, RT-pddex 1454 8710 A41 670, 699-701 7927, 7956-7958 RT-unk A42 4917-4979 12167-12228 Phage A43 4910-4916 12160-12166 Jumbophage A44 5195-5941 12444-13188 Outgroup A45 627-645, 650-659, 7884-7902, 7907- unclassified 693-694, 744, 768- 7916, 7950-7951, 769, 1451, 1455, 8001, 8025-8026, 1467-1468, 1527- 8707, 8711, 8723- 1528, 1570, 1578, 8724, 8782-8783, 1579, 1604-1611, 8824, 8832-8833, 2429-2444, 2583- 8858-8865, 9683- 2591, 2596-2597, 9698, 9837-9845, 2867-2886, 4876- 9850, 10120-10139, 4883, 5144-5146, 12126-12133, 12393- 5152-5176, 5193- 12395, 12401-12425, 5194, 6666-6680, 12442-12443, 13912- 6695-6696, 6702- 13926, 13941-13942, 6787, 6804-6822, 13948-14032, 14049- 6973, 7050-7053, 14067, 14216, 14292- 7134-7257 14295, 14375-14498

Table A provides non-limiting examples of retron reverse transcriptases that may be modified in accordance with the herein methods to obtain a recombinant retron reverse transcriptase or use in the compositions, systems, and methods described herein.

In particular, Table A provides sequence identifiers corresponding to the novel retron RTs identified as a result of the computational discovery work described in the Examples. The table provides sequence identifiers corresponding to the contents of the Sequence Listing included as part of this Specification. The table includes both RT amino acid sequences and RT nucleic acid sequences. The table is organized into forty-five sub-tables each of which represents those sequences forming a single phylogenetic clade of related retron RTs as determined by the computational work described in the Examples.

Table B and Sub-Tables B1-B45: Exemplary ncRNA Sequences Functional Table Seq ID Nos Types B1 16886-17078 I-A B2 17478-17622 I-B1 B3 17677-17756 I-B2 B4 14831-14833, 14838, 14847, 14850-15460 IC B5 N/A ID B6 N/A II B7 17079-17477 II-A1 B8 17660-17676 II-A1 other B9 19031-19080 II-A2 B10 16414-16516 II-A3 B11 17623-17659 II-A4 B12 N/A II-A4 fused B13 19081-19108 II-A5 B14 16397-16413 III B15 16195-16320 III-A1 B16 15779-15925 III-A2 B17 15476-15778 III-A3 B18 16321-16366 III-A4 B19 16367-16396 III-A5 B20 16705-16814 IV B21 16815-16885 V B22 16517-16704 VI B23 N/A VII-A1 B24 N/A VII-A2 B25 N/A VIII B26 18949-19030 IX B27 N/A X B28 14657-14716, 14778-14824, 14834, XI 14835-14836, 14839, 15461-15475 B29 14717-14777, 14841-14846 XII B30 18413-18936 XIII B31 14499-14656 XIV B32 N/A XV B33 18939 XVI B34 N/A XVII B35 N/A CRISPR- associated B36 15926-16178 Ec107-like B37 N/A RT-atpase B38 N/A RT-DUF4116 B39 N/A RT-pddex B40 N/A RT-HTH B41 14837 RT-unk B42 N/A Phage B43 N/A Jumbophage B44 17757-18412 Outgroup B45 14825-14830, 14840, 14848-14849, unclassified 16179-16194, 18937-18938, 18940-18948

Table B provides non-limiting examples of retron ncRNA sequences that may be modified in accordance with the herein methods to obtain a recombinant retron ncRNA sequences for use in the compositions, systems, and methods described herein.

In particular, Table B provides sequence identifiers corresponding to the novel retron RTs identified as a result of the computational discovery work described in the Examples. The table provides sequence identifiers corresponding to the contents of the Sequence Listing included as part of this Specification. The table is organized into forty-five sub-tables each of which represents those sequences forming a single phylogenetic clade of related retron ncRNAs as determined by the computational work described in the Examples.

In various embodiments and in the claims, the disclosure provides recombinant retron-based genome editing systems which comprise combining in a cell through various delivery strategies a retron RT together with an ncDNA. In various aspects, the retron RTs and the ncDNA constituting the recombinant retron-based genome editing system can be based on pairing together such components that are naturally found associated to one another in nature, i.e., sourced from the same bacterial species. These are referred to as the “cognate” pairings of retron RT and retron ncRNA. In various other aspects, the retron RT component and the ncRNA component can be from different bacterial species, i.e., are not found together in nature as cognate pairs. In still other embodiments, the retron RT component and the ncRNA component can both be from the same phylogenetic functional type (e.g., Type I-A, Type I-B1, Type I-B2, Type IC, etc.). For example, a recombinant retron-based genome editing system may be comprised of a retron RT from Type I-A (i.e., SEQ ID Nos: 3980-4178 for AA and SEQ ID Nos: 11231-11429 for NT—see Table A) and a retron ncRNA also from Type I-A (i.e., SEQ ID Nos: 16886-17078—see Table B).

TABLE C Consensus RT amino acid sequence RT Type Consensus Amino Acid Sequence SEQ ID NO: TypeIIA YKVYXIPKRXXGXRXIAXPXXXLKXXQXXXXXXXXXXXXXHXXXXAYXXXXXIKXNAXXHXXXXYXLK 19109 XDXXXFFNSIXXXXXXXXXXXXXXXXXXXXXXXXXXXXFWXXXXXXXXXLXLSXGAPSSPXXSNXXMXXFDXXXXX XCXXXXXXYXRYADDXTFSTXXXXXLXXXPXXXXXXLXXXXXXXXXXNXXKTXFSSKAHNRHXTGXTXXNXXXXSX GRXXKRXIXXLXXXXXXX TypeIIA1 YKXXXIXKXXGXXRXIXXPXXXXKXXQXXXXXXXXXXXXXHXXAXAYXXXXXIXXNAXXHXXXXXXXX 19110 XDFXXFFXSIXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXLXIGXPXSPXXSNXXXXXXDXXXXXX XXXXXXXYXRYADDXXXSXXXXXXXXXXXXXXXXXXXXXXXXXXXXNXXKTXXXXXXXXXXXTGXXXXXXXXXXXG RXXKRXXXXXXXXXXXX TypeIIB1 XXXXXXXXXXXXXXXXXLKXXXXFXXXXXXXXXXXXXXXVXSYRKGXXXXXAVXXHXXXXXFXXXDXX 19111 XFFXSIXXXXXXXXXXXXXXXXPXXDXXXXXXXXXXXXXXXXXLPXGXXTSPXXSNXXLXXFDXXXXXXCXXXXXX YTRYXDDXIXSXXXXXXXXXXXXXXXXXLXXXXXXXXXXNXXKXKXXXXGXXXKXLGXXILPXGXXXXXXXXKXXX EXXXXXXXXX TypeIB2 XXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXSYXXXXXXHXXXXXXXR 19112 XDIXXFFXSIXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXPXGXXXSPXXSNXXFRXXDX XIXXXCXXXXXXYXRYADDXLFSXXXXXXXXXXXXFXXXIXXXXXXXXXXXNXXKXXXXXXXXSLNGXXXXXXXXX XXXSXXKXXXXXXXXXXXXXX TypeIIC YXXFXIXKXXGXXRXIXAPXXXLKXXQXXLXXXLXXXXXXXXXXXXXXXXXXHXFXXXXXIXXNAXXH 19113 XXXXXVXNXDLXXFFXXXXFGRVXGXFXXXXXFXXXXXXAXXXAQXXCXXXXLPQGXPXSPXIXNXIXXXLDXXXX XXAXXXXXXYXRYADDXTFSTXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGFXXNXXKTRXXXXXXRQXVTGLXV NXXXNXXXXYXXXXRXXXXXXXXX TypeIID YXXXXXXKKXGGXRXIXXPXXXLXXXQXXLXXXLXXXYXXXXXXXXXGFXXXXXXXXXXXXIXXNAXX 19114 HXXKXXXLNXDXXXFFXSIXXXXXXXXXXXXXFXXXXXXAXXXXLLXTXXXXLPXGAPXSPXXSNXXCXXXDXXLX XXXXXXXXXYXRYADDLTFSXXXXXXXXXXXXXXXXIXXXXFXXNXKKXRXXXXXXXQXVTGXXVNXKXNXXRXXX XXXRAXXHXXXXX TypeIID YXXXXXXKKXGGXRXIXXPXXXLXXXQXXLXXXLXXXYXXXXXXXXXGFXXXXXXXXXXXXIXXNAXX 19115 HXXKXXXLNXDXXXFFXSIXXXXXXXXXXXXXFXXXXXXAXXXXLLXTXXXXLPXGAPXSPXXSNXXCXXXDXXLX XXXXXXXXXYXRYADDLTFSXXXXXXXXXXXXXXXXIXXXXFXXNXKKXRXXXXXXXQXVTGXXVNXKXNXXRXXX XXXRAXXHXXXXX TypeIIAl YXXXXXXXXXXXXRXXXXPXXXLKXXQXWXXXXXXXXXXXXXXXXAYXXXXSXXXXAXXHXXXXXXXX 19116 XDIXXFFXSXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXLXXGXXXSPXIXNXXMXXXDXXXXXXXXXXX XXYXRYXDDIXXSSXXXIXXXXXXXXXXXLXXXXXXXNXXKTXXXXXXXXXXXTGXXXXXXXXXXGXXXXXXXXXX XYXXXXX TypeIIA2 YRXFXXXKXXGXXRXIXXPRXFXKXXQXXXXDXXLXXLXXHXXXXXXXXXXSXXXNAXXHXXXXXXXX 19117 XDIXXXFXXIXXXXXXXXXXXXXXXXXXXXXXXXXXTXXXXLPQGAPTSPXXSNXXLXXFDXXXXXXXXXXXXXYX RYXDDXTXSXXXXXXXXXXXXXXXXXLXXXXXXXNXXKXRXXXXXXXQXVTGXXXNXXXXPXRXXRXXXRAXXXXA XX TypeIIA3 YXXXXXXKXXXXXRXIXXPXXXLKXXQXWILXXILXXXXXSXXXXXFXXXXXXXXNAXXHXXXXXXLX 19118 XDXXXFFXXXXXXXVXXXFXXXGYXXXXXXXLXXXCXXXXXLPQGXXXSPXXXNLXXXXLDXRXXXXXXXXXXXYT RYADDXXXSXXXXXXXXXXXXXXXXIXXXEXXXXNXXKXXXXXXXXXXXXTGXXXXXXXXXXXXXXXXXRXXXXXX XX TypeIIA4 XXXXXIXXXXXXRKIXTXXXXXXXXXXXHXXXXXXXXXXXXXXXFXKAYXXXXSIXXNAXXHMYNDXF 19119 XXXDIXXFFXXIXHXXLXXXLXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGLXXGXXXSPXLXNXYXKXFDXIXYG XLKXXXXXXXIYTRYADDXXISFKXXXXXXXXXXXIXXXXXXXLXXXXLXXNXXKXXXXXXXXSNHVXITGXXIXX XXXXXRXXXVGXXXXXXLXXXAXXXXXX TypeIIA4 XXXXXXXXXXKXRXXXXXXXXXXGXXXXXXHXXXXXXXXXXXXXXXXSYAYXXXXSIXXCXXXHXXXX 19120 XFXKXDIXXFFNSIXXXXLXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXPXGLXXSPXXSDXYXXXXXXXXXXXX XXXXXXYTRYADDIXISXXXXXXXXXXXXIXXXXXXXLXXXXLXXNXXKXXXXXXXXXXXXXXXXGXNIXXXXXXX XXXVGXXXXXXXXXXXXXXXXX TypeIIA5 YRXFXXXKXXGXXRXIXXPXTYLKVXQWWIXDXIXXXXXXXXXXXGFXXGXXXXXNAXXHXXXXXXLN 19121 XDXXXFFXSXXXXXXXXXFXXXGXXXXXXXXLXXLXXXXXXXPXGAPTSPXXXNXXXXXXDXXLXXXXXXXXXXYX RYADDXTFSXXXXXXXXXXXXXXXXXXXXGFXXXXXKTXXXGXXXRXXVTGXXXNXXXXXXXXXRXXXRXXXHXXX XX TypeIIIA YRXXXIXKXXGXXRXIXEPLPXLKXIQXWILXXILXXXXXSXXAKAXXXXXXXXXNXXXHXXXXXXXX 19122 1 XDXXXFFXXIXXXXXXXXFXXXGYXXXXXXXLXXLCXXXXXLPQGAPTSPXLSNXXXXXXDXXXXXXXXXXXXXYT RYADDXXFSGXXXXXXXXXXXXXXXXXXXXXXNXXKXXXXXXXXXQXVTGXVVNXKXQXXXXXRXXXRXXXXXIXK TypeIIIA YRXFXIXKXXGGXRXIXXPXXXLXXXQXXIXXXILXXXXXXXXXXXXXXXXSXXXNAXXHXXXXXXLK 19123 2 XDXXXFFXSIXXXXXXXXFXXXGYXXXXXXXLXXXCXXXXXLPQGAXTSPXLSNXXXXXXDXXLXXXXXXXXXXYX RYADDXXXSGXXXXXXXXXXXXXXXXXXGXXXNXXKXXXXXXXXXXIXTGXXXXXXXXXXPXXXXRXXXXXXXXXX XX TypeIIIA YXXXXXXKXXXXXRXIXXPXXXLXXXQXXIXXXXLXXXXXHXXXXAXXXXXXXXXXAXXHXXXXWXXK 19124 3 XDXXXFFXXXXEXXXXXXFXXXGYXXLXXXEXARXCTXXXXXXXXXXXXXXXXXXXXXXXXXXXGXLPQGAPTSXX LXNLXXXXXDXXXXXXAXXXXXXYTRYXDDXXXSXXXXXXXXXXXXXXXXXXXXXXXXGXXXXXXKXXXXXPGXXX XVLGLXVXXXXXXLXXXXXXXXXXHXXXXXXX TypeIIIA YXXXXXXKXXGGXRXIXXPXXXLXXXQXWIXXNILXXXXXXXXXXGFXXXXSIXXNAXXHXXXXXXLX 19125 4 XDLXXFXXXIXXXXXXXXFXXXGYXXXXXXXXAXXXTXXXXXXXXXXXXXXXXXXXXLPQGAPXSPXXXNXXXXXX DXRXXXXXXXXXXXYXRYADDXTFSXXXXXXXXXXXXXXIXXXEXXXXNXXKXXXXXXXXXXXVTGLXXXXXXXXX XXXXXXXXXXXXXXCXK TypeIIIA YXXXXIXKXXGXXRXXXXPXXXLKXXQXWILXXILXXXXXXXXXXGFXXXXSIXXNAXXHXXXXXXXX 19126 5 XDXXXFFPXIXXXXXXXXFXXXGYXXXXXXXXXXXCTXXXXLPQGXPXSPXXXNXXXXXXDXRXXXXXXXXXXXYX RYADDXTXSGXXXXXXXXXXXXXIXXXXXXXXNXXKXXXXXXXXXQXVTGXXVNXXXXXXXXXXXXXXXXIYXXXK X TypeIIIu YXXXXXXXXXXXXRXXXXPXXXLKXXQXWIXXNILXXXXXXXXXXXXXXXXSIXXNAXXHXXXXXXXX 19127 nk XDIXXFFXSIXXXXXXXXFXXXXXXXXXXXXXXXXXXXXXXXLXQGXPXSPXXXNXXXXXXDXXXXXXXXXXXXXY XRYADDXXXSXXXXXXXXXXXXXXXXXXXXXXXXNXXKTXXXXXXXXXXXTGXXXXXXXXXXXXXXXXXXXEXXYC XX TypeIIV YXXXXXXKXXGXXRXXXXPXXXXRXXQXRINXRIFXXXXXWPXXXXGSXPXXXXXXXXXXXDYXXCAX 19128 XHCXXKXXLKXDIXXFFXNXXXXXVXXXFXXXXXXXXXXXXXLXXXCXXXXXXXQGXXTSSYXAXLXLXXXEXXXX XXXXXKXLXYTRXVDDITXSSXXXXXXFXXXXXXXXXMLXXXXLPXNXXKXXXXXXXXXXLXVHGLRXXXXXPRXP XXEXXXIRXXVXXXXXX TypeV YXXXXXXXXXXXKXRXXXXPXXXLKXXQKRINXXIFXXXXXPXYLXGGXXXXXXXRDYXXNXXXHXXX 19129 XXXIXLDXXXFYXXIXXXXVXXXXXXXXXFXXXVXXXLXXLXTXXXXXPQGXCTSSYXANLXXXXXEYXXXXXXXX XXXXYXRLLDDXTXSXXXXXXXXXXXXXIXXXXXXXXXXXLXXXXXKXXXXXXXXXXXXXXVTGLWXXXXXPXXXX XXRXXIRXXVXXCXXX TypeVI XXXXXXXXXXXXXRXXXXXXXXLXXXXXXXXXXXXXXXXPXXXXXXXXXXXXXXNAXXHXXXXXXXXX 19130 DXXXFXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXGXXXSXXXXXXXXXXXXXXXXXXXXXXXXXXX XXXDDXXXSXXXXXXXXXXXXXXXXXXXXXXXXXXXKXXXXXXXXXXXXTGXXXXXXXXXXXXXXXXXXXXXXXXX TypeVIIA XXXXXXXXXXXXXXXRXVWEXXXXXXXXXKXXXRXXXXFXXXXXXXXPHXXXXGYXXGRXXRXNAXXH 19131 1 XGXXXXXXXDXXXFFPSIXXXRXXXXXXXXGXXXXXXXXLXXFXTIXXXLPLGLXXSPXXXNXXXXXXDXXLXXLA XXXXXXYXRYXDDXXXSXXXXXPXXXXXXXXXXXXXFXXXXXKXXXSKXGQXHXVTGLSXXXXXXPHXPRXXKXXL RQELXXXXXX TypeVIIA XXXXXXXXXXXXXRXXXXXXXXXXXXXKXXXXXXXXXXXXXXXXGFXXXXXXXXNAXXHXXXXXXXXX 19132 2 DXXXFFXXIXXXXXXXXXXXXXXXXXXXXXXXXXXTXXXXLXXGXXTSPXXXNXXXXXXDXXXXXXXXXXXXXXRY XDDXXFSXXXXXXXXXXXXXXXXXXXXXXXXNXXKXXXXXXGXXQXVTGLXXXXXXXPRXXXXXKXXXRXXXXXXX X TypeVIII YXXXXXXKRXXXXXGEXRXVXXAXXXXXXXXHRXXXXXXXXXXXFGXHVQGFXXXRSXXXNAXXHXXX 19133 XXXXHADIXXFFXXITXXQVXXXXXXXXXXXXXAXXXAXXCTIDGXLXQGTRCSPXXXNXVCXXXDXXXLXLAXXX XXXXXRYADXXTFSGXXXXXXXXXXXXXXXXGFXLRXXXCYXQXXGXXQXVTGLXVXDXXXPRLPKXXKXXLRLXX XXXXKX TypeIX YRXFXIXXXXXXXRXIXAPXVXLKXXQXWXXXXXXXXXXXXXXVXGFXXGXXXXXAAXXHXXAXWXXS 19134 XDXXXFFXXXXXXXXXXXLXXXGYXXXXXXXXXXXXXXXXXLXQGXPXSPXXSNXXXXXXDXXXXXXXXXXXXXXX RYADDXXFSGXXXXXXXXXXXXXXXXXXXXWXXXXXKXXXXXXPXRLKVHGLLVXXXXXXLTKGYRNXXRAXXHXX TypeX YXXXPXXXXXXXXRWIEAPXXXLKXXQRXXLXXXXYXXXXXXXAHGFXXGRSIXXNAXXHXGXXXWX 19135 XDXXXFFPXXXXXXXXXXXXXXXXXXXXXXXXXXLXXXXXXLPQGAPTSPXLXNLVXXXXDXXLXXXAXXXXXXYT RYADDLXFSXXXXXXXXXXXXXXXXXXIXXXXGXXXXXXKXXXXXXXQRQXVTGXVVNXXXXLPXXXRRXLRAXXX XXXXX TypeXI YXXFXIXKXXGXXRXIXXPXXXLKXXQXXXXXXLXXXXXXXXXXXGFXXXXSXXXNAXXHXXXXXXXN 19136 XDLXXFFXXIXXXRXXXXXXXXXXXXXXXXAXXXAXXXXXXXXLPQGAPXSPXXSNXXXXXXDXXLXXXAXXXXXX YTRYADDXTXSXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXXFXXNXXKXXXXXXXXXXXVTGXXXNXXXNXXR XXXXXXXXXXXXXXXX TypeXII YXXFXXXKXXGGXRXIXAPXXXLXXXQXXXXXXXXXXXXXXXXAHGFXXXXSXXXNAXXHXXXXXXXX 19137 XDXXXFFPXXXXXRVXGXFXXXGYXXXXAXXXAXXXTXXXXXXXXXXXXXXXXXXXXRXLPQGAXXSPXXXNXXXX XLDXRLXXXAXXXXXXYTRYADDXTFSXXXXXXXXXXXXXXXXXXXXXXEGFXXXXXKXXXXXXXXXQXVTGXXVN XXXXXXRXXXXXXRAXXXXXXXX TypeXIII YXXFXXPKXXGGXRXIXAPXXXLXXXQXXXLXXXXXXXXXXXXAHGFXXXXSXXXNAXXHXXXXXXXX 19138 XDXXXFFPXXXXXRVXGXFXXXGYXXXXAXXXXLXXTXXXXXXXXXXXXXXXXXXXXRXLPQGAXXSPXXXNXXXX XLDXRLXXXAXXXGXXYTRYADDLTFSXXXXXXXXXXXXXXXXXXXXXXEGFXXXXXKXXXXRXXXXQXVTGXWN XXXXXXRXXXXXXRAXXXXXXXX TypeXIV YXXFXIXKKXGXXRXIXXPXXXLXXXXXXXXXXXXXXXXXXXXXXGFXXXXSXXXNAXXHXXXXXVXN 19139 XDLXDFFXSXXXXRXXXXXXXXPXXXXXXXXAXXXXXLCXXXXXXXXXXXXXLPQGXPXSPXXXNXXCXXLDXXLX XXAXXXXXXYXRYADDXTFSXXXXXXXXXXXFXXXXXXIXXXXXXXXNXXKTRXXXXXXRQEVTGXXVXXXXNVXX XYXXXXRXXLXXWXXX TypeXV YXXFXXXKKSGGXRXIXXPXKSLXIXQXKLSQXLYXXYXPXXXVHGXXXXXSIXTNAXXHXXKXFXLN 19140 XDIXDFFXSINXGRVRGXFIAXPYXLXXXVATXXAXICCXXNKLPQGAPXSPIXSNLICXXXDXELQXFAXXXXXX YTRYADDITXSXXXXXLPXXLXXXXXXXXXXXXLGXELXXIIXXNGFXINXXKXRLXYXXQXQXVTGLXVNXXVNV XRKYIRNXXXXLHAWEKX TypeXVI YXXFXXXKXXGXXRXIXAPXXXLKXXQXXILXXXLXXVXLXXXAXGFRXXRSIXTNAXXHXXXXXXXK 19141 XDXKXFFPSXXXXRVXGXXXXLGYPXXXXXXLTXLXTXXXXLPXGAPTSPXXXNXXXXRXDXRXXXLXXKXXFXYS RYADDXXXSSXXXXXXXXIPFFXXIXXXEGFXXNEXKXXIXRXGXRQXXTGXVVNXKXNXXXXEXXXLRAVXXNCX XX TypeXVII YRXFXXXKXDGXXRXXXXPXXXLKXXQXXXXXXXLXXXXXHPXAXXFXXXXSXXXXAXXHAXXXXXXT 19142 XDXXDFFXXTXXXRVXXXXXXXXXXXXXXXXLXXLXXXXXXLPQGAPTSPXLSNXVNXXXDXXXXXXXXXXXXXYT RYXDDXXFSWXXXXXPXXFXXXXXXXLXXXGYXXXPXKXXXXXXXXXXPXXTGXXLXXXGXXXXPXXXXXXXXXXX X

Table C provides a consensus amino acid sequence for each type of RT identified in Table A.

EXAMPLES Example 1: Retron Engineering

This example demonstrates that an engineered (or recombinant) retron as described herein can be engineered based on existing sequence information in a sequence database.

Specifically, retron-like reverse transcriptases (RTs) are first identified from various genomic or metagenomic sequence databases. The identified ncRNA regions of the retrons are then predicted in silico, and/or are determined empirically by, for example, reconstituting the putative retron system in a living cell, to assay for msDNA production.

Once a particular wild-type retron is identified and confirmed, one or more sequence elements of the retron are modified based on the methods described herein, and/or the associated RT is modified or engineered to enhance the overall activity and/or processivity of the retron. For example, the wild-type retron can be engineered by one or more of the following methods: (a) addition of a heterologous nucleic acid sequence of interest (e.g., a nucleotide sequence encoding an HDR donor template) to the various portions and structures of the msd locus; (b) performing any/all of the structural modifications described herein; (c) optionally linking the engineered retron ncRNA to one or more CRISPR gRNA, e.g., a gRNA linked to the 3′ end of a retron ncRNA, or a gRNA linked to the 5′ end of a retron ncRNA, or a pair of gRNAs linked one to the 3′ end and one to the 5′ end of a retron ncRNA.

The engineered retron or its encoded ncRNA are optionally linked to a sequence-specific nuclease such as CRISPR/Cas enzyme and/or gRNA, ZFN, TALEN, TnpBs, or IscBs, and the like

For example, the RT is fused to the CRISPR/Cas enzyme (such as Cas9 or Cpf1) as a N- or C-terminal fusion, optionally further fused to a nuclear localization signal (NLS) at the N- and/or C-terminal of the fusion. The ncRNA or the resulting msDNA after reverse transcription is also linked to a guide RNA (gRNA) at the 5′ or 3′ end, and can be used with the cognate CRISPR/Cas enzyme in the method of the invention.

In another example, the RT is linked to a DNA-repair modulating biomolecule, such as a HDR promoter and/or an NHEJ peptide inhibitor, as described herein above.

Example 2: Genome Engineering

This example demonstrates that the engineered retron can be used to introduce a heterologous nucleic acid sequence (e.g., a targeting DNA donor or template) into a host cell genome (e.g., a human cell).

First, a targeting DNA is introduced into the msd portion of the engineered retron, as shown in FIG. 3 as a “marker.” The heterologous nucleic acid sequence is designed such that it is flanked by 10-100 or more base pairs of homologous sequence substantially identical/homologous to the genomic sequence at the target site. Thus, the desired edit on the “marker” is in between the homologous sequence arms, and includes an insertion, a deletion, and/or other mutations.

A sequence-specific nuclease, such as a Cas9 nuclease, forms a complex with a guide RNA that specifically targets a location at or near the desired editing site on the genomic sequence. The nuclease is designed such that it does not cut the target once the edit is properly installed. In this experiment, the Cas9 nuclease is linked to the retron reverse transcriptase (RT) as a fusion protein. The Cas9 gRNA is also linked to the ncRNA or msDNA produced by the engineered retron.

The engineered retron encoding the sequence-specific nuclease (and the fused RT) as well as the ncRNA (and the linked gRNA) are then introduced into a host cell, such as a human cell.

The engineered retron is introduced as part of a plasmid for transfection into the cell, or as part of a viral vector (such as an AAV vector) for infecting the cell.

Alternatively, the transcribed ncRNA (and the linked gRNA) can be formulated in vitro in, for example, the lipid nanoparticle or delivery system of the invention, for direct delivery into the host cell. The sequence-specific nuclease (and the fused RT) can either be separately delivered into the host cell (using transfection of plasmid or infection by AAV, etc.), or delivered with the ncRNA in lipid nanoparticles. For example, the coding sequence (e.g., mRNA) of the Cas9-RT fusion is formulated with the ncRNA in the same lipid nanoparticle, or separately formulated as lipid nanoparticles to be delivered together, either simultaneously or one after another.

Once present inside the cell, the Cas9-RT fusion is translated by the host cell translation machinery, while the ncRNA is transcribed from the engineered retron (if the ncRNA is not directly delivered into the cell). The RT portion of the fusion proceeds to reverse transcribe the ncRNA and convert it to msDNA, which includes the heterologous nucleic acid sequence as the cargo/donor/template. Meanwhile, double-stranded breaks (DSBs) are generated at the target site by the CRISPR/Cas9 nuclease. The cargo/donor/template sequence is then integrated into the host genome at the target site, via host cell DNA repair (e.g., HDR) after the Cas9 nuclease forms the DSB.

The cells of interest are then assayed for correct installation of the edit on the heterologous nucleic acid sequence, either through phenotypic changes that occur due to the edit, or by direct DNA sequencing of the target site, or both.

Example 3: Computational Discovery of Novel Retrons and Phylogenetic Analysis to Increase Retron Diversity for Genome Editing Applications

Reverse transcriptases (RTs), also called RNA-directed DNA polymerases, are enzymes capable of synthesizing DNA using RNA as a template. Although they are present in the three domains of life and viruses; prokaryotic RTs have been traditionally less explored in comparison with their eukaryotic counterparts. Prokaryotic RTs can be divided into 6 main groups: (1) Group II introns, (2) CRISPR-associated RTs, (3) Diversity Generating Retroelements, (4) Retrons, (5) Abi (Abortive infection) RTs and (6) Unknown Groups of RTs (UG). In the last five years, a burst of research has increased the knowledge about prokaryotic RTs that led to the discovery of novel putative systems with potential antiphage properties, including retrons. In this Example, a systematic search of public databases was performed with the aim of increasing the number and diversity of known retrons for possible use in genome editing applications. As a result, new types of retrons have been identified, and the increase in data has allowed the identification of new associated ncRNAs.

As a first step in this work, a set of known retron RTs was manually curated, trimmed, and aligned to create suitable input, which was then used to train an HMM model for identifying new retron RTs. Next, this model was applied to existing databases of protein sequences (e.g., nr database from NCBI) to identify potential candidate retron RTs. As a next step, the identified candidates were then grouped by sequence identity and individual representative sequences from each group were chosen. These RT domains of these representative candidates were then aligned, and a phylogenetic tree was built (data not shown). Using information about other known classes of RTs, the full phylogeny was separated into bonafide retron RT candidates and RTs that could belong to other classes (such as group II introns, DGRs, CRISPR-Cas RTs, etc). A new alignment and phylogenetic tree were then constructed from these verified candidates, as shown in FIG. 28 . For all sequences in the new alignment, a matrix of the protein neighborhood was built, indicating which proteins were present in the vicinity of the candidate retron RTs. From there, types and subtypes of retrons were defined based on the RT phylogeny and identity of associated effector proteins.

To predict ncRNAs proximal to the candidate retron RTs, genomic regions proximal to the candidate retron RTs were extracted, aligned, and analyzed for structural covariance using an iterative convergence model.

ncRNA classification methodology:

To identify what type of ncRNA a sequence may belong to, sequences can be checked against the covariance model of existing ncRNA types. The Infernal suite (http://eddylab.org/infernal/) provides tools to do this. Briefly, covariance models can be built from structural alignment of known ncRNAs for each type, then collated into a CM database. From there, new sequences can be searched against the databases to verify whether the sequence fits with any of the represented families.

Example 4: Retron-Mediated Precise Genome Editing: Unique Ability to Produce Donor DNA Template Known as msDNA (Multicopy Single-Stranded DNA) Inside Cells Beyond Current Limit of Insertion Size Using an all-RNA System and in the Presence of Guide RNA Provided in Trans

Current genome editing methods can efficiently disrupt target genes by inserting and deleting genetic information using programmable nucleases. However, gene disruption that does not discriminate mutant from wild type allele impairs not only mutant allele in pathogenesis but also wild type allele in normal physiology of the organism. In Transthyretin (TTR) induced amyloidosis, for example, mutated TTR alleles cause the disease but non-pathogenic wild type allele plays a role in neuro protection and injury response (1). As a single nucleotide exchange is a dominant variant in TTR (2), the vast majority of pathogenic alleles in human differ from their nonpathogenic counterparts by small modifications that require much more precise editing technologies to correct. Homology-directed repair (HDR) stimulated by nuclease-mediated DNA breaks has been widely used to install precise editing. However, HDR relies on the delivery of exogenous donor DNA, that can trigger strong immune response and has been shown inefficient to deliver universally high abundance in recipients (3). Current therapeutical ex-vivo and in vivo delivery of donor DNA relies on AAV (adeno-associated virus) transduction. However, AAV manufacturing is complicated and expensive. Additionally, AAV has raised safety concerns in 30% of 255 complete or ongoing clinical trials surveyed by meta-analysis (4). RNA delivery in contrast, successfully demonstrated its safety, easier manufacturing and efficacy in two SARS-Covid2 vaccines. Based on this amenability of RNA as a cargo, we searched the system, where donor DNA for precise editing could be generated from delivered RNA within cells.

Retrons are defined by their unique ability to produce an unusual satellite DNA known as msDNA (multicopy single-stranded DNA) from RNA inside the cell (5). These bacterial elements are involved in phage defense (6) and consist of a non-coding RNA and a reverse transcriptase (RT) that can reverse transcribe non-coding RNA (ncRNA) into msDNA. Their tightly defined sites of reverse transcription enable to insert donor DNA sequences in non-coding RNA substrate. Moreover, their compact size is amenable to deliver along with programmable nuclease in all RNA format. These features place retron an attractive tool for precise genome editing in therapeutic application.

Independent approach to generate new DNA sequences by reverse transcription in the cell is prime editing and its virus originating RT can achieve up to 44 bp insertion (7). Retron in natural sequence can produce up to 163 nucleotide single strand DNA (8), suggesting that longer insertion in retron mediated editing is feasible. In line with this, newly characterized retron Aco1 could insert 56 bp with 6 bp deletion in EMX1 locus of HEK293T cells in S. pyogenes Cas9 nuclease system.

Evidence of Retron from Different Clades in Precise Editing

As a first step, over 7,000 retron/retron-like RTs in bacterial genomes were identified and analyzed. Phylogenetic analysis of the over 7,000 retrons produced the phylogenetic tree of FIG. 28 Non-coding RNA gene was searched in the genomic neighborhood of the retron/retron-like RT sequences for conserved RNA secondary structures resembling characterized msr-msd transcripts: self-hybridizing inverted repeat at the ends, hairpin structures in msr and msd, such as those represented by FIGS. 2-27 . Using covariance models and consensus structure detection, ncRNAs were identified with a high degree of confidence in certain RTs. To evaluate gene editing applicability of retron across diverse phylogeny, Eco1, Eco3, Eco5, Aco1, RTX3-2042, 6083 v1 and 6943 were selected for further analysis (FIG. 30 ). Eco1, Eco3 and Eco5 were previously experimentally validated retrons to show msDNA production (9). Aco1 was recently annotated in the literature but has not been experimentally validated as producing msDNA (10). RTX3-2042, 6083 v1 and 6943 are novel retrons.

A gene editing assay was first performed in plasmid DNA format. Retron elements are assembled in a plasmid, where RT is transcribed under CAG promoter and ncRNA fused with single guide RNA (sgRNA) of Cas9 nuclease under U6 or H1 RNA polymerase III promoter (see FIGS. 31A and 31B). As described in FIGS. 32 and 33 , the plasmid was transfected to Cas9 expressing human embryonic kidney 293T (HEK293T) cells via lipofection. Three days after transfection, EMX1 target genomic locus was amplified by PCR and sequences were analyzed by next generation sequencing (NGS). The precise edit assayed is characterized as a 10 bp insertion and a 6 bp substitution and its percentage is calculated by CRISPResso 2 analysis among other editing outcomes. A representative editing outcome is shown in FIG. 35 . Eco1 (FIG. 36 ), Aco1 (FIG. 37 ), RTX3-2042 (FIG. 38 ), RTX3-6083 v1 and 6943 (FIG. 39A) showed precise editing efficiency of 0.3%, 0.1%, 0.25%, 0.06% and 0.05% (left graph in FIGS. 36, 37, 38, and 39 , respectively). Undesired editing outcome defined as indels that incorporated random nucleotides or deleted sequences near Cas9 cutting site amounted to 50%, 3%, 5%, 2% and 4% (right graph in FIGS. 36, 37, 38, and 39A, respectively). Follow up experiments using the same assay indicated that RTX3_6083v1 and RTX3_6943 generated 3-4 fold more precise edits than Eco1 while indels generated from these two retrons were 2-3 fold lower (FIGS. 39B and 39C). RTX3_2042 showed precise editing at similar frequencies to Eco1 but had more variability than other samples (FIGS. 39B and 39C). These data demonstrated that recently reported (Aco1) and three novel retrons (RTX3-2042, RTX6083 v1, RTX3-6943) enable precise gene editing in human cells, in addition to previously characterized Eco1 (11).

Evidence of Retron-Mediated Gene Editing in all-RNA System

To establish RNA-based editing system, gene editing assay with two RNA components (RT mRNA and ncRNA fused to sgRNA of Cas9 nuclease) was performed first, in HEK293T cells constitutively expressing Cas9 nuclease. With RT and ncRNA being separated in RNA format, the condition for higher precise editing and lower indels could be easier identified than when they are together in the same plasmid by optimizing the ratio between RT enzyme and its substrate. RT mRNA and ncRNA fused to sgRNA of Cas9 nuclease were produced by in vitro transcription. Following experimental scheme in FIG. 40 . Eco1, Eco3 and Eco5 mediated gene editing activities were compared at two different ratios of RT mRNA and ncRNA-sgRNA fusion in Cas9 expressing HEK293T cells. More amount of ncRNA than RT mRNA was transfected to maximize msDNA production for precise gene editing. Results showed precise edits up to 0.4% for Eco3 and as low as 10% indels for Eco3 (FIG. 41 ). Precise editing frequency in RNA based assay is comparable to that in plasmid-based assay.

Given that Eco3 produced highest editing, RNA load and RT mRNA: ncRNA-sgRNA fusion ratio was further optimized in Eco3 retron system. To 0.2 or 0.5 μg of RT mRNA, ncRNA-sgRNA fusion was added at ratios of RT mRNA to ncRNA, 1:2, 1:3, 1:4, 1:5, 1:8, 1:10, respectively. 0.5 μg of RT mRNA gave rise to more precise editing than 0.2 μg at any ratio and increasing ncRNA correlated with higher precise editing (FIG. 42 , left). Indels was as low as 6% (FIG. 42 , right). With two RNA components, precise editing was achieved up to 1% of cell population.

Next, Cas9 mRNA was added to RT mRNA and ncRNA-sgRNA fusion, making all RNA editing system in HEK293T cells (FIG. 43 ). At the optimized RNA load and ratio of RT mRNA and ncRNA-sgRNA fusion, the amount of Cas9 mRNA was titrated with Eco3 retron (FIG. 44 ). Precise editing was observed up to 0.1% of cell population with 0.2 μg of Cas9 mRNA and adding more it did not increase editing efficiency. While the precise editing frequency is an order of magnitude lower than two RNA system, the editing occurred by specific action of Cas9 nuclease and retron, since the absence of either abrogated the precise editing.

The delivery of all RNA system to cells was also made via lipofection using MessengerMAX reagent (FIG. 45 ). This approach is more resembling in vivo delivery of therapeutic RNAs loaded in lipid nanoparticle (LNP), in terms of formation of RNA/lipid complexes and cell uptake mechanism. When applied to Aco1 retron system that consists of Aco1 RT mRNA, Aco1 ncRNA-sgRNA fusion, and Cas9 mRNA, precise editing of 56 bp insertion with 6 bp deletion was observed in 0.1% of cell population and the frequency of indels was about 1.5% (FIG. 46 ). Aco1 retron was recently annotated but has not been experimentally validated yet (10). The precise gene editing mediated by Aco1 strongly suggest that Aco1 retron could produce msDNA inside cells. Moreover, the length of insertion in precise editing exceeds those made by other reverse transcriptase mediated editing (7).

Elevated Retron-Mediated Gene Editing in all-RNA System with sgRNA Spike-In

In all experiments above, retron ncRNA fused with sgRNA of Cas9 nuclease was used. This fusion RNA serves as a template of RT and at same time sgRNA portion could complex with Cas9 nuclease. Two enzymes acting on a single RNA piece could create steric effects that affect the activity of either enzyme. In line with this hypothesis, frequency of indels that tells overall Cas9 nuclease activity is only about 1.5% with 400 ng of ncRNA-sgRNA fusion (FIG. 46 , right).

To test systematically the hypothesis, Cas9 nuclease activity by frequency of indels was compared between equimolar amounts of ncRNA-sgRNA fusion and separated sgRNA. When electroporated with 200 ng of Cas9 mRNA, 1 μg (7.5 pmol) of ncRNA-sgRNA fusion shows 20-fold lower activity than 0.266 μg (7.5 pmol) of separated sgRNA alone (FIG. 47 ) The result suggests that ncRNA-sgRNA fusion might inhibit either formation of complex with Cas9 protein or the activity of Cas9-sgRNA complex or both. In addition, chemically modified sgRNA shows 6-fold higher activity than unmodified sgRNA. These results indicate that affected Cas9 cleavage activity by ncRNA-sgRNA fusion could limit precise editing and addition of separated sgRNA could compensate for it. Modification of separated sgRNA is expected to further enhance precise editing.

FIG. 48 summarizes the assay of sgRNA spike-in in all RNA system and Fig. R. shows the results using Eco3 retron. At 0.2 μg of Cas9 mRNA and 0.5 μg of RT mRNA, the amount of sgRNA spike-in is titrated at 50, 100, and 200 ng (Fig. R, left). The titration was performed at two different ratios of RT mRNA: ncRNA-sgRNA fusion=1:6 or 1:8. With addition of 50 ng of sgRNA spike-in to 1:6 ratio, the precise editing was achieved 40-fold and gradually increased with more sgRNA, reaching up to 50-fold increase at 200 ng. 1:8 ratio of RT mRNA:ncRNA-sgRNA fusion responded similarly to sgRNA spike-in and obtained slightly higher activity than 1:6, up to 13% of precise editing. Higher precise editing with sgRNA spike-in came along with higher indels on the right graph.

The effect of sgRNA spike-in in precise editing was confirmed using orthogonal delivery method, lipofection (FIG. 50 ). Although overall amount of RNA to transfect was five times lower than in electroporation, 3.4% of precise editing was obtained without sgRNA spike-in, which is 7.5-fold higher than that achieved by electroporation (FIG. 49 and FIG. 51 , left graph). With addition of sgRNA as low as 2 ng, precise editing further increased up to 3.5-fold, reaching 12% efficiency (FIG. 51 , left). Increasing the amount of sgRNA up to 10 ng did not further change the efficiency. The precise editing observed is dependent on retron since the absence of retron components (RT mRNA and ncRNA-sgRNA fusion) completely abrogated the editing.

These data support that physical fusion of ncRNA and sgRNA of Cas9 nuclease could be a limiting factor for Cas9 activity and consequently precise editing and either spike-in of sgRNA demonstrated here or separation of ncRNA and sgRNA would be an improved strategy for efficient precise editing.

Separation of ncRNA and sgRNA

The impact of separating sgRNA from ncRNA on editing was tested in Eco3 retron system comparing the editing efficiency between ncRNA-sgRNA fusion and separated ncRNA and sgRNA side by side (FIG. 52 ). Increasing amount of sgRNA was added to 300 ng of ncRNA that is equimolar amount of 400 ng of ncRNA-sgRNA fusion. With no sgRNA added, no precise editing was observed as expected. With 10 ng of sgRNA, precise editing peaked at 2.23% compared to 1.78% obtained with ncRNA-sgRNA fusion (FIG. 52 , left). The increasing amount of sgRNA beyond 10 ng did not further improve precise editing. Frequency of indels (FIG. 52 , right) showed a similar trend as precise editing.

These data demonstrates that separation of ncRNA and sgRNA could achieve comparable or even higher precise editing than ncRNA-sgRNA fusion.

Materials and Methods:

Mammalian cell culture HEK293T (ATCC CRF-3216) or 293T-Cas9 (Genecopoeia SL502) cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus GlutaMAX (Thermo Fisher Scientitic), supplemented with 10% (v/v) fetal bovine serum (Gibco). Cells were maintained at 37′C with 5% CO2.

Genomic DNA extraction After incubation, the media was removed from the cells and genomic DNA was extracted by the addition of prepGEM reagent (Thomas Scientific: PUN0050) directly into each well of the tissue culture plate. Lysed cells were transferred to a 96 well PCR plate and incubated at 72° C. for 10 minutes, followed by 95° C. enzyme inactivation step for 2 minutes.

High-throughput DNA sequencing of genomic DNA samples Human EMX1 gene locus was amplified from genomic DNA samples and sequenced on an Illumina NextSeq. Briefly, amplification primers containing Illumina forward and reverse adapters were used for a first round of PCR (PCR1) amplifying EMX1 targeting site. 25 μl PCR1 reactions were performed with 0.3 μM of each forward and reverse primer, 1 μl of genomic DNA extract and 12.5 μl of KAPA HIFI HOTSTART PCR master mix. PCR reactions were carried out as follows: 95° C. for 1 minute, then 25 cycles of [98° C. for 20 seconds, 65° C. for 15 seconds, and 72′C for 15 seconds], followed by a final 72° C. extension for 2 minutes. PCR reactions were purified using Ampure XP beads (Beckman Coulter) and eluted in 20 μl H20. Unique Illumina barcoding primer pairs were added to each sample in a secondary PCR reaction (PCR2). Specifically, 25 μl PCR2 reactions were performed with 5 μl of IDT for Illumina UDI primers (Illumina), 1 μl of purified PCR1 reaction, and 12.5 μl of KAPA HIFI HOTSTART PCR master mix. PCR reactions were carried out as follows: 95° C. for 3 minutes, then 10 cycles of [95° C. for 30 seconds, 55° C. for 30 seconds, and 72′C for 30 seconds], followed by a final 72° C. extension for 5 minutes. PCR2 reactions were purified by SequalPrep Normalization plate kit (Thermo Fisher Scientific) and pooled. Size and purity were evaluated by Tape station D1000 assay (Agilent). DNA concentration was measured by fluorometric quantification (Qubit, ThermoFisher Scientific) and libraries were sequenced with 30% PhiX sequencing control on an Illumina NextSeq 2000 instrument using P1 or P2 300 cycle kit. Sequencing reads were demultiplexed and alignment of amplicon sequences to a reference sequence was performed using CRISPresso2. CRISPresso2 was run in HDR mode using the desired allele as the expected allele and precise editing yield was calculated as the number of HDR aligned reads divided by total aligned reads.

Plasmid based gene editing Plasmids encoding retrons were synthesized by Twist Bioscience. For transfections, 25,000 293T-Cas9 cells (Genecopoeia: SL502) were transfected using Lipofectamine 3000 (Thermofisher: L3000001) according to the manufacturer's protocol. Transfected cells were incubated at 37° C. for 72 hrs before lysis in prepGEM reagent (Thomas Scientific: PUN0050) according to the manufacturer's protocol. A 1 uL aliquot of crude lysate was used as template for amplification of the EMX1 CRISPR target site by PCR. Illumina adaptors and UDIs were added in a second round of PCR before loading onto an Illumina NextSeq. The resulting FASTQ files were used as input for CRISPResso2 which quantified precise editing and indels at the EMX1 locus.

In vitro transcription Plasmid templates were synthesized by Twist Bioscience. T7 promoter was inserted upstream of RT or ncRNA sequences. Plasmid was linearized by Sbfl-HF enzyme (NEB, R3642S) for 16 hour at 37′C and the linearization was checked by agarose gel electrophoresis. RT mRNA was synthesized for 16 hour at 37′C using HiScribe T7 mRNA kit with CleanCap Reagent AG (NEB, E2080S) and ncRNA using HiScribe T7 High Yield RNA synthesis kit (NEB, 2040S). After DNAse1 treatment, RNA was purified using Qiagen RNeasy midi kit (Qiagen, 75144). RNA concentration was measured by Nanodrop (Thermo Fisher) and purity and size were evaluated by RNA ScreenTape analysis on Tape station (Agilent). RT mRNA was further poly (A) tailed by E. Coli Poly(A) polymerase (NEB, M0276) and purified by Qiagen RNeasy midi kit.

RNA Transfection by Neon Electroporation system HEK293T (ATCC CRF-3216) or 293T-Cas9 (Genecopoeia SL502) cells were harvested and resuspended in R buffer. 50,000 cells with 1 μl of RNA mixture were electroporated by 10 μl Neon tip at 1150 voltage, 20 pulses. Electroporated cells were gently transferred to 96 well plate (VWR) containing culture media and cultured for 72 hours before genomic DNA extraction.

RNA Transfection by Lipofectamine MessengerMAX 20,000 HEK293T (ATCC CRF-3216) cells were seeded in 96 well plates. 16-24 hours post-seeding, cells were transfected with 0.3 μl of Lipofectamine MessengerMax (Thermo Fisher Scientific) with appropriate amount of RNA mixture. Cells were cultured for 72 hours before genomic DNA extraction.

Sequences Utilized in Example 4 EMX1 s.p Cas9 gRNA GAGTCCGAGCAGAAGAAGAA Eco1 ncRNA + HDR_Template TGATAAGATTCCGTATGCGCACCCTTAGCGAGAGG TTTATCATTAAGGTCAACCTCTGGATGTTGTTTCG GCATCCTGCATTGAATCTGAGTTACTGTCTGTTTT CCTACAAACGGCAGAAGCTGGAGGAGGAAGGGCCT GAGTCCGAGCAGAAGAAAAAGTTCTCCCATCACAT CAACCGGTGGCGCATTGCCACGAAGCAGGCCAATG GGGAGGACATCGATGTCAAGGAAACCCGTTTCTTC TGACGTAAGGGTGCGCAT ACGGAATCTTATCA Aco1 ncRNA + HDR Template CCGTAGTGGGAGCCTCAGGCGAGGGTGTGTATCAT GCCCGTTCTGCCAAGACCCACCAAAGAAGGGCACC GTGGAGGACAAACGGCAGAAGCTGGAGGAGGAAGG GCCTGAGTCCGAGCAGAAGAAAAAGTTCTCCCATC ACATCAACCGGTGGCGCATTGCCACGAAGCAGGCC AATGGGGAGGACATCGATGTCACCTCCACGGTGC AATGCGAAAGCAACTTGAGGCTT TGCTTAGTATGAGGCTCCCACTACGG RTX3_2042 RT MKDDQYSQWKKYYESRGILPEIQDKLLNYAKIHID NNTPVIFNFEHLTLLLGREKNYLSSVVNSPDSHYR KFKIKKRSGGEREITAPYLSLLEMQYWIYRNILIN VKIHYAAHGFAQDKSIITNSRNHLGQKHLLKMDLK DFFPSIKLNRIIYIFKSLGYPNIIAFYLASICSYK GHLPQGSPTSPILSNIVSITLDNRLVKFARKMKLR YSRYADDLTFSGDKIPTNYIKYITDIINDEGFEVN DTKTKLYLKAGKRIVTGISVIGNDPKLPREYKRKL KQELHYIFTYGIGSHMAKKKIKKINYLYRII GKVNFWLNIEPDNEYARNAKAKLLLLIDN* RTX3_2042 ncRNA + HDR_Template GTTAAGGTGGTTATATTCTAGTATTTATGAAGTGT AGTCGCTTCGATCGTTAAGGCTGATTTTAACCTCT GCATAATAATATCGGTAGATATTATTATGCACGCT CCCTTTAGCAGAGCTAAGAATCGCTCACTCAGGCA CAAGCTTTGAGGACAAACGGCAGAAGCTGGAGGAG GAAGGGCCTGAGTCCGAGCAGAAGAAAAAGTTCTC CCATCACATCAACCGGTGGCGCATTGCCACGAAGC AGGCCAATGGGGAGGACATCGATGTCACCTCAAAG CTTGTGCCTGAGTGAGAGCTAAAGAAA AGAAAAGTAGAATAAGCCACCTTAAC RTX3_6083v1 RT MSNPQPTRAEIFERIKQSSKQEVILEEMQRLGFWP RSEGQPEVAADLIQREGELQRELAELNKKLAVKRN PERALREMRKQRMKDARDKREVTKRAQAQQRYDKA LLWHEKRASHVAYLGPGVSASLHENSSATQEQGDK GKPKRARDRAVPDLQRLTLNGLPALISAAQLAESM GVSVAELRFLSFHREVARTNHYHSFTLPKKTGGER LISAPMPRLKRAQYWVLDNVLAKMPAHDAAHGFLA GRSIISNAKPHAGQDVVINLDVKDFFPSIAFGRIK GVFRQLGYGESIATVFALLCSENRAQAWQVDGERL FVGGKARERVLPQGAPTSPMLTNLLCRRMDRRLLG LAKQLGFVYTRYADDLTFSASGEPARDNVGKLLSR VRWILRDEGFTPHPDKERVMRKGRRQEVTGLVVNS DTPSVSRETRRRLRAALHRASQPDAASKPAHWQGH TAQPSQLLGLATFVHQIDPKQGKTLLADAQQLMRS PIDRANDAAKSASRADAAQQSFRVLAAAGKPPVLA DGKNWWQPAPPATPVLEKTDQQRREERQATRRQQA AAAAPPPSSTRRNERPQQAAHEQQGDAQPQNEAPP RFDPDQYAPPPRNVMTYWAQIAISFFLGSILHNRL ITIFAMVAVIALYYMRRQRWDVFMGILVVATLLGY LVRGMG* RTX3_6083v1 ncRNA + HDR Template GCTCCGGAGCAATGAGCAGGCTCTTGCAATCCGGG CGGTGTTTCGCCGCCCTTGTGAACTGCCGTTTCAT GCACCACGGGCGCCGTTTTCACTGTGCCACCCCAG CCACGGTAGTGCTACAAACGGCAGAAGCTGGAGGA GGAAGGGCCTGAGTCCGAGCAGAAGAAAAAGTTCT CCCATCACATCAACCGGTGGCGCATTGCCACGAAG CAGGCCAATGGGGAGGACATCGATGTCAGGGGAGG ACATCGATGTCAAGCACTACCGTGGCGG GGTGGCGTCGAGCGAACAGCTCCCGTCCCGTGAGC CCTACAGGCTCTTCGACGAGATGCACATTGCTCCG GAGC RTX3_6943 RT MEESTNYKLLVWGLSVIQPATPNEVLNYLTSTLND NGLLPDVEKMIHYFELLDQLGYIHQVSKRNNLYSL TPRGNERLTPALKRLRDKIRLFMLDNCHSISKLGV LASTDTENMGGDSPSLQLRHNLKEVPHPSLSWAAG TLPSSPRQAWVRIYEQLNIGSMSSDEASTPTTARN APLSFVGRLGFSLNYYSFNKIDEPLFNNDGVTAIA SCIGISPGLITAMVKSPKRYYRTFNLRKKSGGFRS ILAPRKFIKTIQYWLKDHVLNRLKIHSSCYSYRSG VSIKDNAINHVKKKFVASIDISDYFGSINKKMVKD CFYKNNIPDHIVNTISGIVTYNDVLPQGAPTSPII SNAILFEFDEEMTAHALTLDCIYTRYSDDISISSD YKENIAILINIAEANLLSAGFTLNRQKQRIASDNS RQVVTGILVNESIRPTRCYRKKIRSAFDHALKEQD GSQLTINKLRGYLNYLKSFETYGFKFNEKKYKETL DFLIALKQS* RTX3_6943 ncRNA + HDR Template GCGGAGTGCTGGCCTCAACTGATACAGAGAATATG GGCGGTGATTCGCCGTCTTTACAGTTAAGGCACAA TTTAAAAGAGGTTCCGCACCCAAGCCTGTCTTGGG CTGCACAAACGGCAGAAGCTGGAGGAGGAAGGGCC TGAGTCCGAGCAGAAGAAAAAGTTCTCCCATCACA TCAACCGGTGGCGCATTGCCACGAAGCAGGCCAAT GGGGAGGACATCGATGTCAGCAGCCCAAGACAGGC TTGGGTGCGGATCTACGAG CAATTAAATATTGGTTCGATGTCCAGTGATGAGGC CAGCACCCGC Eco1 RT Coding sequence only. RNA was produced from a CRO and proprietary 5′ cap, 5′ UTR, 3′ UTR and encoded polyA of around 120 bases were added. Full substitution with IN-methyl- pseudouridine) AUGAAAUCUGCAGAGUAUCUGAAUACGUUCCGCCU UAGGAAUUUGGGCCUCCCCGUGAUGAACAAUCUCC ACGAUAUGAGCAAGGCGACUCGAAUAUCCGUGGAA ACGCUGAGACUGCUCAUCUAUACAGCAGACUUUCG GUACAGGAUCUACACGGUCGAAAAGAAGGGGCCUG AGAAACGCAUGCGAACAAUUUAUCAACCUAGCCGA GAGCUCAAGGCGUUGCAGGGCUGGGUUCUUCGAAA CAUCCUUGACAAACUCUCAUCAUCACCCUUUAGUA UUGGGUUUGAAAAGCACCAAAGCAUCCUUAACAAC GCGACGCCACACAUAGGUGCCAAUUUCAUAUUGAA CAUCGACUUGGAGGAUUUUUUUCCGAGCCUCACAG CCAAUAAAGUGUUCGGUGUUUUUCACAGUCUUGGG UACAAUCGCCUUAUUAGUUCCGUUCUUACCAAGAU UUGUUGUUACAAGAAUCUCUUGCCCCAGGGAGCAC CCAGCAGUCCGAAAUUGGCGAAUUUGAUUUGUUCC AAGCUCGAUUAUCGAAUACAAGGGUACGCGGGCAG CCGGGGACUCAUCUAUACCCGCUACGCAGACGAUC UUACGCUGUCUGCCCAAUCAAUGAAGAAGGUCGUA AAGGCGCGGGAUUUCUUGUUUUCUAUCAUCCCGUC CGAGGGCUUGGUAAUUAAUUCCAAAAAGACUUGUA UCUCAGGACCACGAUCUCAGCGAAAAGUGACAGGA CUCGUCAUUUCUCAAGAAAAAGUCGGUAUAGGGAG AGAGAAGUAUAAGGAAAUCCGCGCGAAGAUCCACC ACAUAUUCUGUGGCAAGAGCAGCGAGAUAGAACAC GUCCGAGGCUGGUUGUCCUUCAUACUGAGCGUGGA CUCAAAAAGCCACCGCCGGUUGAUCACCUAUAUUU CAAAACUGGAAAAGAAAUAUGGAAAGAACCCACUC AACAAAGCUAAAACACCACCAAAGAAGAAAAGAAA GGUCUGA Eco1 ncRNA ncRNA-sgRNA fusion (6 bp substition in EMX1 gene) GAAAUGAUAAGAUUCCGUAUGCGCACCCUUAGCGA GAGGUUUAUCAUUAAGGUCAACCUCUGGAUGUUGU UUCGGCAUCCUGCAUUGAAUCUGAGUUACUGUCUG UUUUCCUACAAACGGCAGAAGCUGGAGGAGGAAGG GCCUGAGUCCGAGCAGAAGAAAAAGUUCUCCCAUC ACAUCAACCGGUGGCGCAUUGCCACGAAGCAGGCC AAUGGGGAGGACAUCGAUGUCAAGGAAACCCGUUU CUUCUGACGUAAGGGUGCGCAUACGGAAUCUUAUC AGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUG CUGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGU CCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGU GCUUUUUUUUUUUU Eco3 RT Coding sequence only. RNA was produced from a CRO and proprietary 5′ cap, 5′ UTR, 3′ UTR and encoded polyA of around 120 bases were added. Full substitution with IN-methyl- pseudouridine AUGCGCAUUUACUCUCUGAUCGACAGCCAAACCUU AAUGACCAAAGGGUUCGCAUCCGAGGUCAUGAGGA GCCCAGAACCCCCUAAGAAGUGGGACAUUGCGAAG AAGAAGGGCGGAAUGCGUACGAUAUACCAUCCCUC UUCUAAGGUGAAGCUGAUACAGUACUGGCUGAUGA ACAACGUGUUCUCCAAAUUGCCGAUGCACAACGCC GCGUACGCUUUCGUGAAGAAUAGAUCUAUCAAGUC UAACGCACUGCUGCACGCAGAGAGUAAGAACAAAU ACUACGUUAAGAUUGACCUGAAGGACUUCUUUCCA AGCAUCAAGUUCACAGACUUCGAAUAUGCCUUUAC CCGGUACCGUGACAGAAUAGAGUUCACGACCGAGU ACGACAAAGAACUGCUUCAGCUGAUUAAGACCAUU UGUUUCAUUUCUGACUCUACACUGCCAAUAGGCUU CCCCACUUCCCCUCUUAUAGCCAAUUUCGUCGCCA GGGAGCUGGACGAGAAGCUCACUCAGAAGCUGAAC GCUAUAGACAAGCUCAACGCUACGUACACUCGCUA CGCAGACGACAUAAUCGUGAGCACGAACAUGAAGG GCGCCUCUAAGCUGAUCUUAGACUGCUUCAAGCGG ACCAUGAAGGAAAUCGGACCCGAUUUCAAGAUCAA UAUCAAGAAGUUCAAAAUAUGCUCUGCCAGUGGCG GCUCAAUUGUCGUGACGGGCCUUAAGGUCUGUCAU GACUUCCACAUAACUCUGCACCGGUCUAUGAAGGA CAAGAUCCGCCUGCACCUCUCUCUCCUGUCCAAAG GUAUUCUGAAGGACGAGGACCACAACAAGCUGUCC GGGUACAUCGCCUACGCUAAGGACAUCGAUCCACA CUUCUACACCAAGCUCAAUAGGAAGUACUUCCAGG AGAUCAAGUGGAUACAAAACCUGCAUAAUAAGGUG GAGCCACCAAAGAAGAAAAGAAAGGUCUGA Eco3 ncRNA ncRNA-sgRNA fusion (10 bp insertion in EMX1 gene) GAAAUGAUAAGAUUCCGUAAGAGCCAAACCUAGCA UUUUAUGGGUUAAUAGCCCAUCGGGCCAUGAGUCA UGGUUUCGCCUAGUAUUUUAGCUAUGCCCGUCGUU CAGUUCGCUGAACAAACGGCAGAAGCUGGAGGAGG AAGGGCCUGAGUCCGAGCAGAAGAAUUACGUCUGC AAAGUUCUCCCAUCACAUCAACCGGUGGCGCAUUG CCACGAAGCAGGCCAAUGGGGAGGACAUCGAUGUC AUCAGCGAACUGAUCGACGUGCUCAAGUAGGUUUG GCUCUUACGGAAUCUUAUCAGAGUCCGAGCAGAAG AAGAAGUUUCAGAGCUAUGCUGGAAACAGCAUAGC AAGUUGAAAUAAGGCUAGUCCGUUAUCAACUUGAA AAAGUGGCACCGAGUCGGUGCUUUUUUUUUUUU Eco5 RT Coding sequence only. RNA was produced from a CRO and proprietary 5′ cap, 5′ UTR, 3′ UTR and encoded polyA of around 120 bases were added. Full substitution with IN-methyl- pseudouridine AUGGACGCUACCAGAACGACUCUCCUUGCAUUGGA UCUCUUCGGAUCUCCAGGUUGGUCCGCCGAUAAAG AAAUUCAGAGGCUUCAUGCGCUCAGUAAUCAUGCU GGAAGGCAUUACAGAAGGAUUAUAUUAAGUAAAAG GCACGGCGGACAGCGUCUUGUGCUUGCACCUGAUU ACUUGUUAAAGACCGUUCAGCGCAACAUUUUGAAG AACGUUUUGAGUCAAUUUCCACUGUCACCAUUUGC UACAGCCUACAGACCGGGAUGCCCAAUCGUGUCUA ACGCGCAGCCACACUGCCAACAGCCACAGAUCUUG AAACUCGAUAUAGAAAACUUCUUCGAUUCUAUUAG UUGGUUGCAGGUGUGGCGGGUGUUUCGCCAGGCCC AGUUGCCCCGAAAUGUCGUAACGAUGCUCACUUGG AUAUGUUGUUAUAACGACGCACUUCCGCAGGGUGC CCCUACAUCCCCUGCAAUUUCCAAUCUCGUCAUGA GAAGGUUUGAUGAACGGAUUGGAGAAUGGUGUCAG GCUCGAGGGAUUACCUACACUCGCUACUGCGAUGA CAUGACGUUUAGUGGACACUUCAAUGCAAGGCAGG UCAAGAAUAAAGUCUGCGGUCUCUUAGCUGAGCUG GGCCUUUCCCUGAAUAAACGGAAAGGCUGCCUCAU AGCGGCUUGUAAGCGCCAGCAAGUCACCGGCAUUG UUGUGAAUCACAAGCCACAGCUUGCCCGAGAAGCC AGGCGUGCCCUGCGUCAGGAAGUGCACCUGUGCCA GAAAUAUGGAGUUAUCUCUCAUCUCUCACAUAGAG GUGAACUGGAUCCUAGCGGAGAUCUGCACGCUCAG GCGACAGCGUAUCUCUAUGCACUCCAGGGGAGAAU UAACUGGCUUCUUCAAAUUAACCCUGAGGAUGAGG CGUUUCAACAGGCCCGGGAGUCCGUUAAGAGGAUG UUAGUUGCCUGGCCACCAAAGAAGAAAAGAAAGGU CUGA Eco5 ncRNA ncRNA-sgRNA fusion (10 bp insertion in EMX1 gene) GAAAUGAUAAGAUUCCGUACGCCAGCAGUGGCAAU AGCGUUUCCGGCCUUUUGUGCCGGGAGGGUCGGCG AGUCGCUGACUUAACGCCAGUAGUAUGUCCAUAUA CCCAAAGUCGCUUCAUUGUAACAAACGGCAGAAGC UGGAGGAGGAAGGGCCUGAGUCCGAGCAGAAGAAU CCUUCGAGCAAAGUUCUCCCAUCACAUCAACCGGU GGCGCAUUGCCACGAAGCAGGCCAAUGGGGAGGAC AUCGAUGUCAUACAGUUACGCGCCUUCGGGAUGGU UUAAUGGUAUUGCCGCUGUUGGCGUACGGAAUCUU AUCAGAGUCCGAGCAGAAGAAGAAGUUUCAGAGCU AUGCUGGAAACAGCAUAGCAAGUUGAAAUAAGGCU AGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUC GGUGCUUUUUUUUUUUU EMX1 sgRNA modified Contains chemical modifications from Synthego: 2′-F, 2′-O-methyl, phosphorothioates GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGC UGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUC CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG CUUUUU EMX1 sgRNA unmodified No modifications GAGUCCGAGCAGAAGAAGAAGUUUCAGAGCUAUGC UGGAAACAGCAUAGCAAGUUGAAAUAAGGCUAGUC CGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUG CUUUUU

REFERENCES

-   [1] Are we creating a new phenotype? MF, Dohrn. 2021, Neurological     Research and Practice. -   [2] Val50Met hereditary transthyretin amyloidosis:not just a medical     problem but a psychosocial burden. J, Gonzalez-Moreno. s.l.:     Orphanet Journal of Rare Diseases, 2021, Vol. 16. -   [3] synthetic DNA delivery systems. D, Luo. 2000, Nature     Biotechnology, pp. 33-37. -   [4] rAAV immunogenicity, toxicity, and durability in 255 clinical     trials: A meta-analysis. W, Shen. 2022, Frontiers in Immunology. -   [5] Reverse transcriptase associated with the biosynthesis of the     branched RNA-linked msDNA in myxococcus xanthus. S, inouye. s.l.:     Cell, 1989, Vol. 56. 709-717. -   [9] Bacterial retrons function in anti-phage defense. A, Millman.     s.l.: Cell, 2020, Vol. 183. 1551-1561. -   [7] Search-and-replace genome editing without double-strand breaks     or donor DNA. AV, anzalone. 2019, Nature, pp. 149-157. -   [8] Branched RNA covalently linked to the 5′ end of a     single-stranded DNA in Stigmatella aurantiaca. T, Furuichi. 1, s.l.:     Cell, 1987, Vol. 48. 47-53. -   [9] Retrons and their applications in genome engineering. A J,     Simon. 21, s.l.: Nucleic Acids Research, 2019, Vol. 47. -   [10] Systematic prediction of genes functionally associated with     bacterial retrons and classification of the encoded tripartite     system. MR, Mestre. 22, s.l.: Nucleic Acids Research, 2020, Vol. 48. -   [11] Precise genome editing across kingdoms of life using     retron-derived DNA. SC, lopez. 2, s.l.: Nature Chemical Biology,     2021, Vol. 18.

Example 5: Improved ncRNAs Generated by Transcription from Template Linearized Plasmid Having Blunt Ends Show Higher Precise Editing Efficiency

Previously made in vitro transcription experiments to produce ncRNA used a double-stranded DNA template containing a 3′ overhang (on same strand as T7 promoter sequence). A new template with a blunt end was designed and tested. See FIG. 52. As shown in FIG. 53 , four RNAs (cas9 mRNA, Eco3 RT mRNA, gRNA targeting EMX1 locus and ncRNA) were transfected into 293T cells using MessengerMAX lipofection reagent. Cells were harvested after 3 days, genomic DNA isolated, the EMX1 locus amplified, the Illumina libraries produced and sequenced on NextSeq. The percent of precise edits improves more than 5-fold for ncRNA produced using blunt end template as compared to the precise editing by ncRNA produced using overhang template.

Example 6: Improved ncRNAs with MS2 Hairpin

Additional elements can be added to the ncRNA by encoding them on the plasmid template. FIG. 53 shows a modified Eco3 ncRNA that comprises an MS2 stem loop hairpin structure added to its 3′ end. As shown in FIG. 54 , the precise editing of ncRNA comprising the 3′ MS2 structure resulted in nearly 15-fold increase in precise editing compared to an ncRNA generated from a overhang template, and nearly 3-fold increase in precise editing compared to an ncRNA generated from a blunt end template.

Example 7: Modified ncRNAs with Caps and/or Tails

ncRNA performance can be modified by adding a cap structure to the 5′ end and/or adding a poly(A) tail at the 3′ end, as depicted in FIG. 55 . As shown in FIG. 56 , using ncRNA with either or both protection by cap and tail lowered indels as compared uncapped/untailed ncRNAs. In this experiment, a 4 component all-RNA system (RT mRNA+Cas9 mRNA+ncRNA-sgRNA+sgRNA) was delivered to HEK293T cells by Lipofectamine MessengerMAX. All RNA was transfected at a fixed amount RT mRNA 100 ng, ncRNA-sgRNA 400 ng, Cas9 mRNA 100 ng, and sgRNA 5 ng. ncRNA-gRNA fusion was either capped (+cap −tail) or poly-A tailed (−cap +tail) or both capped and poly-A tailed (+cap +tail). Using RNA without end protection (−cap −tail) produced ˜4.5% precise edits and the editing was dependent on retron since the absence of RT abrogated precise editing. Using RNA with either or both protection by cap and tail produced lower precise editing (left graph) but lowered indels (right graph) than without cap and tail. 

1. A gene editing system comprising one or more delivery vehicles, wherein: the delivery vehicle(s) comprise RNA cargo, said RNA cargo comprises (a) at least one mRNA molecule encoding (i) a nucleic acid programmable nuclease and (ii) a retron reverse transcriptase, (b) an engineered retron ncRNA, and (c) guide RNA for the programmable nuclease, each delivery vehicle contains (a)(i) and/or (a)(ii) and/or (b) and/or (c), whereby one delivery vehicle or more than one delivery vehicle delivers (a)(i), (a)(ii), (b), and (c).
 2. The gene editing system of claim 1, wherein (a)(i) and (a)(ii) comprise a single mRNA molecule encoding the nucleic acid programmable nuclease and the retron reverse transcriptase.
 3. The gene editing system of claim 2, wherein (a)(i) and (a)(ii) are encoded and expressed as a fusion protein.
 4. The gene editing system of claim 3, wherein the fusion protein comprises the C-terminal end of the nucleic acid programmable nuclease fused to the N-terminal end of the retron reverse transcriptase (nuclease:RT fusion).
 5. The gene editing system of claim 3, wherein the fusion protein comprises the N-terminal end of the nucleic acid programmable nuclease fused to the C-terminal end of the retron reverse transcriptase (RT:nuclease fusion).
 6. The gene editing system of claim 1, wherein (a)(i) and (a)(ii) comprise a first mRNA molecule encoding the nucleic acid programmable nuclease and a second mRNA molecule encoding the retron reverse transcriptase.
 7. The gene editing system of claim 1, wherein (c) is separate from (a)(i), (a)(ii) and (b) or is provided in trans.
 8. The gene editing system of claim 1, wherein (b) the engineered retron ncRNA, and (c) the guide RNA are fused or are provided in cis.
 9. The gene editing system of claim 8, wherein the guide RNA is fused to the 5′ end of the retron ncRNA.
 10. The gene editing system of claim 8, wherein the guide RNA is fused to the 3′ end of the retron ncRNA.
 11. The gene editing system of claim 8, wherein the engineered ncRNA comprises a first guide RNA fused to the 5′ end of the retron ncRNA, and a second guide RNA fused to the 3′ end of the retron ncRNA, and the first and second guide RNAs target different sequences.
 12. The gene editing system of claim 1, wherein the one or more delivery vehicles comprise a liposome or a lipid nanoparticle (LNP).
 13. The gene editing system of claim 1, wherein (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retron reverse transcriptase, and (b) the engineered retron ncRNA are in the same delivery vehicle.
 14. The gene editing system of claim 1, wherein the (a) the at least one mRNA molecule encoding (i) the nucleic acid programmable nuclease and (ii) the retron reverse transcriptase, and (b) the engineered retron ncRNA are in separate delivery vehicles.
 15. The gene editing system of claim 1, wherein the nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in the same delivery vehicle.
 16. The gene editing system of claim 1, wherein the nucleic acid programmable nuclease and the retron reverse transcriptase are encoded on separate mRNA molecules and those separate mRNA molecules of (a)(i) and (a)(ii) are contained in different delivery vehicles.
 17. The gene editing system of claim 1, wherein the engineered retron ncRNA includes a sequence of interest encoding a donor polynucleotide comprising an intended edit to be integrated at a target sequence in a cell, and wherein the donor polynucleotide is flanked by a 5′ homology arm that hybridizes to a sequence 5′ to the target sequence and a 3′ homology arm that hybridizes to a sequence 3′ to the target sequence.
 18. The gene editing system of claim 1, wherein the nucleic acid programmable nuclease comprises a Cas9 nuclease, a TnpB nuclease, or a Cas12a nuclease.
 19. The gene editing system of claim 18, wherein the nucleic acid programmable nuclease comprises a Cas9 nuclease.
 20. The gene editing system of claim 1, wherein the retron reverse transcriptase has an amino acid sequence of Table A, or an amino acid sequence having at least 90%, 95%, 99%, or 100% sequence identity with any amino acid sequence of Table A.
 21. The gene editing system of claim 1, wherein the engineered retron ncRNA comprises: A) a pre-msr sequence having a first complementary region of the retron ncRNA; B) an msr sequence including an msr stem-loop structure; C) an msd sequence including an msd stem-loop structure and a sequence of interest, wherein said msd sequence templates a single strand DNA product (RT-DNA) in the presence of the retron reverse transcriptase; and D) a post-msd sequence having a second complementary region, wherein the first and second complementary regions form an a1/a2 duplex region of the retron ncRNA, wherein the msr stem-loop structure, the msd stem-loop structure, or the a1/a2 duplex comprise a modification which result in increased editing efficiency in the presence of a nucleic acid programmable nuclease that associates with the one or more guide RNAs, and wherein optionally one or more of the guide RNAs of (c) are coupled to the pre-msr sequence, the post-msd sequence, or both the pre-msr sequence and the post-msd sequence.
 22. The gene editing system of claim 21, wherein the sequence of interest encodes a donor polynucleotide comprising an intended edit to be integrated at a target sequence of a cell, wherein the donor polynucleotide is flanked by a 5′ homology arm that hybridizes to a sequence 5′ to the target sequence and a 3′ homology arm that hybridizes to a sequence 3′ to the target sequence.
 23. The gene editing system of claim 21, wherein the ncRNA has a nucleotide sequence of any sequences of Table B, or a nucleotide sequence having at least 90%, 95%, 99%, or 100% sequence identity with any sequence of Table B.
 24. An isolated cell comprising the gene editing system of claim
 1. 25. The isolated cell of claim 24, wherein the isolated cell is a mammalian cell.
 26. The isolated cell of claim 25, wherein the mammalian cell is a human cell.
 27. A composition comprising: a) the gene editing system of claim 1; and b) a pharmaceutically or veterinarily acceptable carrier.
 28. The composition of claim 27, wherein the delivery vehicle is a lipid nanoparticle comprising: a) one or more ionizable lipids; b) one or more structural lipids; c) one or more PEGylated lipids; and d) one or more phospholipids.
 29. The composition of claim 28, wherein the one or more ionizable lipids comprises an ionizable lipid set forth in Table
 2. 30. A method of genetically modifying a cell comprising: contacting the gene editing system of claim 17 with the cell, thereby delivering the RNA cargo to the cell, wherein: the nucleic acid programmable nuclease forms a complex with the guide RNA, wherein said guide RNA directs the complex to the target sequence, the nucleic acid programmable nuclease creates a double-stranded break in in the target sequence, the retron reverse transcriptase and engineered retron ncRNA create RT DNA that comprises the donor polynucleotide, and the donor polynucleotide becomes integrated at the target sequence. 